Arrays of proteins and methods of use thereof

ABSTRACT

Protein arrays for the parallel, in vitro screening of biomolecular activity are provided. Methods of using the protein arrays are also disclosed. On the arrays, a plurality of different proteins, such as different members of a single protein family, are immobilized on one or more organic thinfims on the substrate surface. The protein arrays are particularly useful in drug development, proteomics, and clinical diagnostics.

[0001] This application is a continuation of co-pending application Ser.No. 09/353,215, filed Jul. 14, 1999, which is a continuation-in-part ofco-pending application Ser. No. 09/115,455, filed Jul. 14, 1998, both ofwhich are incorporated herein by reference in their entirety for allpurposes and the specific purposes disclosed throughout thisapplication.

BACKGROUND OF THE INVENTION

[0002] a) Field of the Invention

[0003] The present invention relates generally to arrays of proteins andmethods for the parallel in vitro screening of a plurality ofprotein-analyte interactions. More specifically, the present inventionrelates to uses of the arrays for drug development, proteomics, andclinical diagnostics.

[0004] b) Description of Related Art

[0005] A vast number of new drug targets are now being identified usinga combination of genomics, bioinformatics, genetics, and high-throughputbiochemistry. Genomics provides information on the genetic compositionand the activity of an organism's genes. Bioinformatics uses computeralgorithms to recognize and predict structural patterns in DNA andproteins, defining families of related genes and proteins. Theinformation gained from the combination of these approaches is expectedto greatly boost the number of drug targets (usually, proteins).

[0006] The number of chemical compounds available for screening aspotential drugs is also growing dramatically due to recent advances incombinatorial chemistry, the production of large numbers of organiccompounds through rapid parallel and automated synthesis. The compoundsproduced in the combinatorial libraries being generated will faroutnumber those compounds being prepared by traditional, manual means,natural product extracts, or those in the historical compound files oflarge pharmaceutical companies.

[0007] Both the rapid increase of new drug targets and the availabilityof vast libraries of chemical compounds creates an enormous demand fornew technologies which improve the screening process. Currenttechnological approaches which attempt to address this need includemultiwell-plate based screening systems, cell-based screening systems,microfluidics-based screening systems, and screening of soluble targetsagainst solid-phase synthesized drug components.

[0008] Automated multiwell formats are the best developedhigh-throughput screening systems. Automated 96-well plate-basedscreening systems are the most widely used. The current trend in platebased screening systems is to reduce the volume of the reaction wellsfurther, thereby increasing the density of the wells per plate (96-wellto 384- and 1536-well per plate). The reduction in reaction volumesresults in increased throughput, dramatically decreased bioreagentcosts, and a decrease in the number of plates which need to be managedby automation.

[0009] However, although increases in well numbers per plate aredesirable for high throughput efficiency, the use of volumes smallerthan 1 microliter in the well format generates significant problems withevaporation, dispensing times, protein inactivation, and assayadaptation. Proteins are very sensitive to the physical and chemicalproperties of the reaction chamber surfaces. Proteins are prone todenaturation at the liquid/solid and liquid/air interfaces.Miniaturization of assays to volumes smaller than 1 microliter increasesthe surface to volume ratio substantially. (Changing volumes from 1microliter to 10 nanoliter increases the surface ratio by 460%, leadingto increased protein inactivation.) Furthermore, solutions ofsubmicroliter volumes evaporate rapidly, within seconds to a fewminutes, when in contact with air. Maintaining microscopic volumes inopen systems is therefore very difficult.

[0010] Other types of high-throughput assays, such as miniaturizedcell-based assays are also being developed. Miniaturized cell-basedassays have the potential to generate screening data of superior qualityand accuracy, due to their in vivo nature. However, the interaction ofdrug compounds with proteins other than the desired targets is a seriousproblem related to this approach which leads to a high rate of falsepositive results.

[0011] Microfluidics-based screening systems that measure in vitroreactions in solution make use of ten to several-hundred micrometer widechannels. Micropumps, electroosmotic flow, integrated valves and mixingdevices control liquid movement through the channel network.Microfluidic networks prevent evaporation but, due to the large surfaceto volume ratio, result in significant protein inactivation. Thesuccessful use of microfluidic networks in biomolecule screening remainsto be shown.

[0012] Drug screening of soluble targets against solid-phase synthesizeddrug components is intrinsically limited. The surfaces required forsolid state organic synthesis are chemically diverse and often cause theinactivation or non-specific binding of proteins, leading to a high rateof false-positive results. Furthermore, the chemical diversity of drugcompounds is limited by the combinatorial synthesis approach that isused to generate the compounds at the interface. Another majordisadvantage of this approach stems from the limited accessibility ofthe binding site of the soluble target protein to the immobilized drugcandidates.

[0013] Miniaturized DNA chip technologies have been developed (forexample, see U.S. Pat. Nos. 5,412,087, 5,445,934 and 5,744,305) and arecurrently being exploited for nucleic acid hybridization assays.However, DNA biochip technology is not transferable to protein arraysbecause the chemistries and materials used for DNA biochips are notreadily transferable to use with proteins. Nucleic acids withstandtemperatures up to 100° C., can be dried and re-hydrated without loss ofactivity, and can be bound directly to organic adhesion layers supportedby materials such as glass while maintaining their activity. Incontrast, proteins must remain hydrated, kept at ambient temperatures,and are very sensitive to the physical and chemical properties of thesupport materials. Therefore, maintaining protein activity at theliquid-solid interface requires entirely different immobilizationstrategies than those used for nucleic acids. Additionally, the properorientation of the protein at the interface is desirable to ensureaccessibility of their active sites with interacting molecules. Withminiaturization of the chip and decreased feature sizes the ratio ofaccessible to non-accessible antibodies becomes increasingly relevantand important.

[0014] In addition to the goal of achieving high-throughput screening ofcompounds against targets to identify potential drug leads, researchersalso need to be able to identify highly specific lead compounds early inthe drug discovery process. Analyzing a multitude of members of aprotein family or forms of a polymorphic protein in parallel(multitarget screening) enables quick identification of highly specificlead compounds. Proteins within a structural family share similarbinding sites and catalytic mechanisms. Often, a compound thateffectively interferes with the activity of one family member alsointerferes with other members of the same family. Using standardtechnology to discover such additional interactions requires atremendous effort in time and costs and as a consequence is simply notdone.

[0015] However, cross-reactivity of a drug with related proteins can bethe cause of low efficacy or even side effects in patients. Forinstance, AZT, a major treatment for AIDS, blocks not only viralpolymerases, but also human polymerases, causing deleterious sideeffects. Cross-reactivity with closely related proteins is also aproblem with nonsteroidal anti-inflammatory drugs (NSAIDs) and aspirin.These drugs inhibit cyclooxygenase-2, an enzyme which promotes pain andinflammation. However, the same drugs also strongly inhibit a relatedenzyme, cyclooxygenase-1, that is responsible for keeping the stomachlining and kidneys healthy, leading to common side-effects includingstomach irritation.

[0016] For the foregoing reasons, there is a need for miniaturizedprotein arrays and for methods for the parallel, in vitro, screening ofthe interactions between a plurality of proteins and one or moreanalytes in a manner that minimizes reagent volumes and proteininactivation problems.

SUMMARY OF THE INVENTION

[0017] The present invention is directed to miniaturized protein arraysand methods of use thereof that satisfy the need for parallel, in vitro,screening of the interactions between a plurality of proteins and one ormore analytes in a manner that minimizes reagent Volumes and proteininactivation problems.

[0018] In one embodiment, the present invention provides an array ofproteins which comprises a substrate, at least one organic thinfilm onsome or all of the substrate surface, and a plurality of patchesarranged in discrete, known regions on portions of the substrate surfacecovered by organic thinfilm, wherein each of said patches comprises aprotein immobilized on the underlying organic thinflm. Preferably, aplurality of different proteins are present on separate patches of thearray.

[0019] In a second embodiment, the invention provides a method forscreening a plurality of proteins for their ability to interact with acomponent of a sample. The method of this embodiment comprisesdelivering the sample to the array of proteins of the invention, anddetecting, either directly or indirectly, for the interaction of thecomponent with the immobilized protein of each patch.

[0020] In a third embodiment, the invention provides a method forscreening a plurality of proteins for their ability to bind a particularcomponent of a sample. The method of this embodiment comprises firstdelivering the sample to the array of proteins of the invention. In afinal step, the method comprises detecting, either directly orindirectly, for the presence or amount of the particular component whichis retained at each patch. Optionally, the method comprises theadditional step of further characterizing the particular componentretained at the site of at least one patch.

[0021] In an alternative embodiment, the invention provides a method ofassaying for protein-protein binding interactions. The first step of themethod of this embodiment comprises delivering a sample comprising atleast one protein to be assayed for binding to the protein array of theinvention. The last step comprises detecting, either directly orindirectly, for the presence or amount of the protein from the samplewhich is retained at each patch.

[0022] In another embodiment of the invention, a method for assaying fora plurality of analytes in a sample is provided which comprisesdelivering the sample to a protein array of the invention and detectingfor the interaction of the analytes with the immobilized protein at eachpatch.

[0023] In still another embodiment of the invention, an alternativemethod for assaying for a plurality of analytes in a sample is providedwhich comprises delivering the fluid sample to a protein array of theinvention and detecting either directly or indirectly, for the presenceor amount of analyte retained at each patch.

BRIEF DESCRIPTION OF THE FIGURES

[0024]FIG. 1 shows the top view of an array of protein-reactive patches.

[0025]FIG. 2 shows the cross section of an individual patch of the arrayof FIG. 1.

[0026]FIG. 3 shows the cross section of a row of monolayer-coveredpatches of the array of FIG. 1.

[0027]FIG. 4 shows a thiolreactive monolayer on a substrate.

[0028]FIG. 5 shows an aminoreactive monolayer on a coated substrate.

[0029]FIG. 6 shows the immobilization of a protein on a monolayer-coatedsubstrate via an affinity tag.

[0030]FIG. 7 shows the immobilization of a protein on a monolayer-coatedsubstrate via an affinity tag and an adaptor.

[0031]FIG. 8 shows a schematic of a fluorescence detection unit whichmay be used to monitor interaction of the proteins of the array with ananalyte.

[0032]FIG. 9 shows a schematic of an ellipsometric detection unit whichmay be used to monitor interactions between analytes and the proteins ofthe array.

DETAILED DESCRIPTION OF THE INVENTION

[0033] A variety of protein arrays, methods, and protein-coatedsubstrates useful for drug development, proteomics, clinicaldiagnostics, and related applications are provided by the presentinvention.

[0034] (a) Definitions

[0035] A “protein” means a polymer of amino acid residues linkedtogether by peptide bonds. The term, as used herein, refers to proteins,polypeptides, and peptides of any size, structure, or function.Typically, however, a protein will be at least six amino acids long.Preferably, if the protein is a short peptide, it will be at least about10 amino acid residues long. A protein may be naturally occurring,recombinant, or synthetic, or any combination of these. A protein mayalso be just a fragment of a naturally occurring protein or peptide. Aprotein may be a single molecule or may be a multi-molecular complex.The term protein may also apply to amino acid polymers in which one ormore amino acid residues is an artificial chemical analogue of acorresponding naturally occurring amino acid. An amino acid polymer inwhich one or more amino acid residues is an “unnatural” amino acid, notcorresponding to any naturally occurring amino acid, is also encompassedby the use of the term “protein” herein.

[0036] A “fragment of a protein” means a protein which is a portion ofanother protein.

[0037] For instance, fragments of a proteins may be polypeptidesobtained by digesting full-length protein isolated from cultured cells.A fragment of a protein will typically comprise at least six aminoacids. More typically, the fragment will comprise at least ten aminoacids. Preferably, the fragment comprises at least about 16 amino acids.

[0038] The term “antibody” means an immunoglobulin, whether natural orwholly or partially synthetically produced. All derivatives thereofwhich maintain specific binding ability are also included in the term.The term also covers any protein having a binding domain which ishomologous or largely homologous to an immunoglobulin binding domain.These proteins may be derived from natural sources, or partly or whollysynthetically produced. An antibody may be monoclonal or polyclonal. Theantibody may be a member of any immunoglobulin class, including any ofthe human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgGclass, however, are preferred in the present invention.

[0039] The term “antibody fragment” refers to any derivative of anantibody which is less than full-length. Preferably, the antibodyfragment retains at least a significant portion of the full-lengthantibody's specific binding ability. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv, dsFvdiabody, and Fd fragments. The antibody fragment may be produced by anymeans. For instance, the antibody fragment may be enzymatically orchemically produced by fragmentation of an intact antibody or it may berecombinantly produced from a gene encoding the partial antibodysequence. Alternatively, the antibody fragment may be wholly orpartially synthetically produced. The antibody fragment may optionallybe a single chain antibody fragment. Alternatively, the fragment maycomprise multiple chains which are linked together, for instance, bydisulfide linkages. The fragment may also optionally be a multimolecularcomplex. A functional antibody fragment will typically comprise at leastabout 50 amino acids and more typically will comprise at least about 200amino acids.

[0040] Single-chain Fvs (scFvs) are recombinant antibody fragmentsconsisting of only the variable light chain (V_(L)) and variable heavychain (V_(H)) covalently connected to one another by a polypeptidelinker. Either V_(L) or V_(H) may be the NH₂-terminal domain. Thepolypeptide linker may be of variable length and composition so long asthe two variable domains are bridged without serious stericinterference. Typically, the linkers are comprised primarily ofstretches of glycine and serine residues with some glutamic acid orlysine residues interspersed for solubility.

[0041] “Diabodies” are dimeric scFvs. The components of diabodiestypically have shorter peptide linkers than most scFvs and they show apreference for associating as dimers.

[0042] An “Fv” fragment is an antibody fragment which consists of one VHand one VL domain held together by noncovalent interactions. The term“dsFv” is used herein to refer to an Fv with an engineeredintermolecular disulfide bond to stabilize the V_(H)-V_(L) pair.

[0043] A “F(ab′)₂” fragment is an antibody fragment essentiallyequivalent to that obtained from immunoglobulins (typically IgG) bydigestion with an enzyme pepsin at pH 4.04.5. The fragment may berecombinantly produced.

[0044] A “Fab′” fragment is an antibody fragment essentially equivalentto that obtained by reduction of the disulfide bridge or bridges joiningthe two heavy chain pieces in the F(ab′)₂ fragment. The Fab′ fragmentmay be recombinantly produced.

[0045] A “Fab” fragment is an antibody fragment essentially equivalentto that obtained by digestion of immunoglobulins (typically IgG) withthe enzyme papain. The Fab fragment may be recombinandy produced. Theheavy chain segment of the Fab fragment is the Fd piece.

[0046] The term “protein-capture agent” means a molecule or amulti-molecular complex which can bind a protein to itself.Protein-capture agents preferably bind their binding partners in asubstantially specific manner. Protein-capture agents with adissociation constant (K_(D)) of less than about 10⁻⁶ are preferred.Antibodies or antibody fragments are highly suitable as protein-captureagents. Antigens may also serve as protein-capture agents, since theyare capable of binding antibodies. A receptor which binds a proteinligand is another example of a possible protein-capture agent.Protein-capture agents are understood not to be limited to agents whichonly interact with their binding partners through noncovalentinteractions. Protein-capture agents may also optionally becomecovalently attached to the proteins which they bind. For instance, theprotein-capture agent may be photocrosslinked to its binding partnerfollowing binding.

[0047] The term “binding partner” means a protein which is bound by aparticular protein-capture agent, preferably in a substantially specificmanner. In some cases, the binding partner may be the protein normallybound in vivo by a protein which is a protein-capture agent. In otherembodiments, however, the binding partner may be the protein or peptideon which the protein-capture agent was selected (through in vitro or invivo selection) or raised (as in the case of antibodies). A bindingpartner may be shared by more than one protein-capture agent. Forinstance, a binding partner which is bound by a variety of polyclonalantibodies may bear a number of different epitopes. One protein-captureagent may also bind to a multitude of binding partners (for instance, ifthe binding partners share the. same epitope),

[0048] “Conditions suitable for protein binding” means those conditions(in terms of salt concentration, pH, detergent, protein concentration,temperature, etc.) which allow for binding to occur between a proteinand its binding partner in solution. Preferably, the conditions are notso lenient that a significant amount of nonspecific protein bindingoccurs.

[0049] A “body fluid” may be any liquid substance extracted, excreted,or secreted from an organism or tissue of an organism. The body fluidneed not necessarily contain cells. Body fluids of relevance to thepresent invention include, but are not limited to, whole blood, serum,urine, plasma, cerebral spinal fluid, tears, sinovial fluid, andamniotic fluid.

[0050] An “array” is an arrangement of entities in a pattern on asubstrate. Although the pattern is typically a two-dimensional pattern,the pattern may also be a three-dimensional pattern.

[0051] The term “substrate” refers to the bulk, underlying, and corematerial of the arrays of the invention.

[0052] The terms “micromachining” and “microfabrication” both refer toany number of techniques which are useful in the generation ofmicrostructures (structures with feature sizes of sub-millimeter scale).Such technologies include, but are not limited to, laser ablation,electrodeposition, physical and chemical vapor deposition,photolithography, and wet chemical and dry etching. Related technologiessuch as injection molding and LIGA (x-ray lithography,electrodeposition, and molding) are also included. Most of thesetechniques were originally developed for use in semiconductors,microelectronics, and Micro-ElectroMechanical Systems (MEMS) but areapplicable to the present invention as well.

[0053] The term “coating” means a layer that is either naturally orsynthetically formed on or applied to the surface of the substrate. Forinstance, exposure of a substrate, such as silicon, to air results inoxidation of the exposed surface. In the case of a substrate made ofsilicon, a silicon oxide coating is formed on the surface upon exposureto air. In other instances, the coating is not derived from thesubstrate and may be placed upon the surface via mechanical, physical,electrical, or chemical means. An example of this type of coating wouldbe a metal coating that is applied to a silicon or polymer substrate ora silicon nitride coating that is applied to a silicon substrate.Although a coating may be of any thickness, typically the coating has athickness smaller than that of the substrate.

[0054] An “interlayer” is an additional coating or layer that ispositioned between the first coating and the substrate. Multipleinterlayers may optionally be used together. The primary purpose of atypical interlayer is to aid adhesion between the first coating and thesubstrate. One such example is the use of a titanium or chromiuminterlayer to help adhere a gold coating to a silicon or glass surface.However, other possible functions of an interlayer are also anticipated.For instance, some interlayers may perform a role in the detectionsystem of the array (such as a semiconductor or metal layer between anonconductive substrate and a nonconductive coating).

[0055] An “organic thinfilm” is a thin layer of organic molecules whichhas been applied to a substrate or to a coating on a substrate ifpresent. Typically, an organic ini is less than about 20 nm thick.Optionally, an organic thinfilm may be less than about 10 nm thick. Anorganic thinfilm may be disordered or ordered. For instance, an organicthinfim can be amorphous (such as a chemisorbed or spin-coated polymer)or highly organized (such as a Langmuir-Blodgett film or self-assembledmonolayer). An organic thinfilm may be heterogeneous or homogeneous.Organic thinfilms which are monolayers are preferred. A lipid bilayer ormonolayer is a preferred organic thinfilm. Optionally, the organicthinfilm may comprise a combination of more than one form of organicthinfilm. For instance, an organic thinfilm may comprise a lipid bilayeron top of a self-assembled monolayer. A hydrogel may also compose anorganic thinfilm. The organic thinfilm will typically havefunctionalities exposed on its surface which serve to enhance thesurface conditions of a substrate or the coating on a substrate in anyof a number of ways. For instance, exposed functionalities of theorganic thinfilm are typically useful in the binding or covalentimmobilization of the proteins to the patches of the array.Alternatively, the organic thinflim may bear functional groups (such aspolyethylene glycol (PEG)) which reduce the non-specific binding ofmolecules to the surface. Other exposed functionalities serve to tetherthe thinfilm to the surface of the substrate or the coating. Particularfunctionalities of the organic thinfilm may also be designed to enablecertain detection techniques to be used with the surface. Alternatively,the organic thinfilm may serve the purpose of preventing inactivation ofa protein immobilized on a patch of the array or analytes which areproteins from occurring upon contact with the surface of a substrate ora coating on the surface of a substrate.

[0056] A “monolayer” is a single-molecule thick organic thinfilm. Amonolayer may be disordered or ordered. A monolayer may optionally be apolymeric compound, such as a polynonionic polymer, a polyionic polymer,or a block-copolymer. For instance, the monolayer may be composed of apoly(amino acid) such as polylysine. A monolayer which is aself-assembled monolayer, however, is most preferred. One face of theself-assembled monolayer is typically composed of chemicalfunctionalities on the termini of the organic molecules that arechemisorbed or physisorbed onto the surface of the substrate or, ifpresent, the coating on the substrate. Examples of suitablefunctionalities of monolayers include the positively charged aminogroups of poly-L-lysine for use on negatively charged surfaces andthiols for use on gold surfaces. Typically, the other face of theself-assembled monolayer is exposed and may bear any number of chemicalfunctionalities (end groups). Preferably, the molecules of theself-assembled monolayer are highly ordered.

[0057] A “self-assembled monolayer” is a monolayer which is created bythe spontaneous assembly of molecules. The self-assembled monolayer maybe ordered, disordered, or exhibit short- to long-range order.

[0058] An “affinity tag” is a functional moiety capable of directly orindirectly immobilizing a protein onto an exposed functionality of theorganic thinfilm. Preferably, the affinity tag enables the site-specificimmobilization and thus enhances orientation of the protein onto theorganic thinfilm. In some cases, the affinity tag may be a simplechemical functional group. Other possibilities include amino acids,poly(amino acid) tags, or full-length proteins. Still otherpossibilities include carbohydrates and nucleic acids. For instance, theaffinity tag may be a polynucleotide which hybridizes to anotherpolynucleotide serving as a functional group on the organic thinfilm oranother polynucleotide serving as an adaptor. The affinity tag may alsobe a synthetic chemical moiety. If the organic thinfilm of each of thepatches comprises a lipid bilayer or monolayer, then a membrane anchoris a suitable affinity tag. The affinity tag may be covalently ornoncovalently attached to the protein. For instance, if the affinity tagis covalently attached to the protein it may be attached via chemicalconjugation or as a fusion protein. The affinity tag may also beattached to the protein via a cleavable linkage. Alternatively, theaffinity tag may not be directly in contact with the protein. Theaffinity tag may instead be separated from the protein by an adaptor.The affinity tag may immobilize the protein to the organic thinfilmeither through noncovalent interactions or through a covalent linkage.

[0059] An “adaptor”, for purposes of this invention, is any entity thatlinks an affinity tag to the immobilized protein of a patch of thearray. The adaptor may be, but need not necessarily be, a discretemolecule that is noncovalently attached to both the affinity tag and theprotein. The adaptor can instead be covalently attached to the affinitytag or the protein or both (via chemical conjugation or as a fusionprotein, for instance). Proteins such as full-length proteins,polypeptides, or peptides are typical adaptors. Other possible adaptorsinclude carbohydrates and nucleic acids.

[0060] The term “fusion protein” refers to a protein composed of two ormore polypeptides that, although typically unjoined in their nativestate, are joined by their respective amino and carboxyl termini througha peptide linkage to form a single continuous polypeptide. It isunderstood that the two or more polypeptide components can either bedirectly joined or indirectly joined through a peptide linker/spacer.

[0061] The term “normal physiological condition” means conditions thatare typical inside a living organism or a cell. While it is recognizedthat some organs or organisms provide extreme conditions, theintra-organismal and intra-cellular environment normally varies aroundpH 7 (i.e., from pH 6.5 to pH 7.5), contains water as the predominantsolvent, and exists at a temperature above 0° C. and below 50° C. Itwill be recognized that the concentration of various salts depends onthe organ, organism, cell, or cellular compartment used as a reference.

[0062] “Proteomics” means the study of or the characterization of eitherthe proteome or some fraction of the proteome. The “proteome” is thetotal collection of the intracellular proteins of a cell or populationof cells and the proteins secreted by the cell or population of cells.This characterization most typically includes measurements of thepresence, and usually quantity, of the proteins which have beenexpressed by a cell. The function, structural characteristics (such aspost translational modification), and location within the cell of theproteins may also be studied. “Functional proteomics” refers to thestudy of the functional characteristics, activity level, and structuralcharacteristics of the protein expression products of a cell orpopulation of cells.

[0063] (b) Arrays of Proteins.

[0064] The present invention is directed to arrays of proteins.Typically, the protein arrays comprise micrometer-scale, two-dimensionalpatterns of patches of proteins immobilized on an organic thinfilmcoating on the surface of the substrate.

[0065] In one embodiment, the present invention provides an array ofproteins which comprises a substrate, at least one organic thinfilm onsome or all of the substrate surface, and a plurality of patchesarranged in discrete, known regions on portions of the substrate surfacecovered by organic thinfilm, wherein each of said patches comprises aprotein immobilized on the underlying organic thinfilm.

[0066] In most cases, the array will comprise at least about tenpatches. In a preferred embodiment, the array comprises at least about50 patches. In a particularly preferred embodiment the array comprisesat least about 100 patches. In alternative preferred Embodiments, thearray of proteins may comprise more than 10³, 10⁴ or 10⁵ patches.

[0067] The area of surface of the substrate covered by each of thepatches is preferably no more than about 0.25 mm². Preferably, the areaof the substrate surface covered by each of the patches is between about1 μm² and about 10,000 μm². In a particularly preferred embodiment, eachpatch covers an area of the substrate surface from about 100 μm² toabout 2,500 μm². In an alternative embodiment, a patch on the array maycover an area of the substrate surface as small as about 2,500 nm²,although patches of such small size are generally not necessary for theuse of the array.

[0068] The patches of the array may be of any geometric shape. Forinstance, the patches may be rectangular or circular. The patches of thearray may also be irregularly shaped.

[0069] The distance separating the patches of the array can vary.Preferably, the patches of the array are separated from neighboringpatches by about 1 μm to about 500 μm. Typically, the distanceseparating the patches is roughly proportional to the diameter or sidelength of the patches on the array if the patches have dimensionsgreater than about 10 μm. If the patch size is smaller, then thedistance separating the patches will typically be larger than thedimensions of the patch.

[0070] In a preferred embodiment of the array, the patches of the arrayare all contained within an area of about 1 cm² or less on the surfaceof the substrate. In one preferred embodiment of the array, therefore,the array comprises 100 or more patches within a total area of about 1cm² or less on the surface of the substrate. Alternatively, aparticularly preferred array comprises 10³ or more patches within atotal area of about 1 cm² or less. A preferred array may even optionallycomprise 10⁴ or 10⁵ or more patches within an area of about 1 cm² orless on the surface of the substrate. In other embodiments of theinvention, all of the patches of the array are contained within an areaof about 1 mm² or less on the surface of the substrate.

[0071] Typically, only one type of protein is immobilized on each patchof the array. In a preferred embodiment of the array, the proteinimmobilized on one patch differs from the protein immobilized on asecond patch of the same array. In such an embodiment, a plurality ofdifferent proteins are present on separate patches of the array.Typically the array comprises at least about ten different proteins.Preferably, the array comprises at least about 50 different proteins.More preferably, the array comprises at least about 100 differentproteins. Alternative preferred arrays comprise more than about 10³different proteins or more than about 10⁴ different proteins. The arraymay even optionally comprise more than about 10⁵ different proteins.

[0072] In one embodiment of the array, each of the patches of the arraycomprises a different protein. For instance, an array comprising about100 patches could comprise about 100 different proteins. Likewise, anarray of about 10,000 patches could comprise about 10,000 differentproteins. In an alternative embodiment, however, each different proteinis immobilized on more than one separate patch on the array. Forinstance, each different protein may optionally be present on two to sixdifferent patches. An array of the invention, therefore, may compriseabout three-thousand protein patches, but only comprise about onethousand different proteins since each different protein is present onthree different patches.

[0073] In another embodiment of the present invention, although theprotein of one patch is different from that of another, the proteins arerelated. In a preferred embodiment, the two different proteins aremembers of the same protein family. The different proteins on theinvention array may be either functionally related or just suspected ofbeing functionally related. In another embodiment of the inventionarray, however, the function of the immobilized proteins may be unknown.In this case, the different proteins on the different patches of thearray share a similarity in structure or sequence or are simplysuspected of sharing a similarity in structure or sequence.Alternatively, the immobilized proteins may be just fragments ofdifferent members of a protein family.

[0074] The proteins immobilized on the array of the invention may bemembers of a protein family such as a receptor family (examples: growthfactor receptors, catecholamine receptors, amino acid derivativereceptors, cytokine receptors, lectins), ligand family (examples:cytokines, serpins), enzyme family (examples: proteases, kinases,phosphatases, ras-like GTPases, hydrolases), and transcription factors(examples: steroid hormone receptors, heat-shock transcription factors,zinc-finger proteins, leucinezipper proteins, homeodomain proteins). Inone embodiment, the different immobilized proteins are all HIV proteasesor hepatitis C virus (HCV) proteases. In other embodiments of theinvention, the immobilized proteins on the patches of the array are allhormone receptors, neurotransmitter receptors, extracellular matrixreceptors, antibodies, DNA-binding proteins, intracellular signaltransduction modulators and effectors, apoptosis-related factors, DNAsynthesis factors, DNA repair factors, DNA recombination factors, orcell-surface antigens.

[0075] In a preferred embodiment, the protein immobilized on each patchis an antibody or antibody fragment. The antibodies or antibodyfragments of the array may optionally be single-chain Fvs, Fabfragments, Fab′ fragments, F(ab′)₂ fragments, Fv fragments, dsFvsdiabodies, Fd fragments, full-length, antigen-specific polyclonalantibodies, or full-length monoclonal antibodies. In a preferredembodiment, the immobilized proteins on the patches of the array aremonoclonal antibodies, Fab fragments or single-chain Fvs.

[0076] In another preferred embodiment of the invention, the proteinsimmobilized to each patch of the array are protein-capture agents.

[0077] In an alternative embodiment of the invention array, the proteinson different patches are identical.

[0078] Biosensors, micromachined devices, and diagnostic devices thatcomprise the protein arrays of the invention are also contemplated bythe present invention.

[0079] (c) Substrates, Coating, and Organic Thinfilms.

[0080] The substrate of the array may be either organic or inorganic,biological or non-biological, or any combination of these materials. Inone embodiment, the substrate is transparent or translucent. The portionof the surface of the substrate on which the patches reside ispreferably flat and firm or semi-film. However, the array of the presentinvention need not necessarily be flat or entirely two-dimensional.Significant topological features may be present on the surface of thesubstrate surrounding the patches, between the patches or beneath thepatches. For instance, walls or other barriers may separate the patchesof the array.

[0081] Numerous materials are suitable for use as a substrate in thearray embodiment of the invention. For instance, the substrate of theinvention array can comprise a material selected from a group consistingof silicon, silica, quartz, glass, controlled pore glass, carbon,alumina, titania, tantalum oxide, germanium, silicon nitride, zeolites,and gallium arsenide. Many metals such as gold, platinum, aluminum,copper, titanium, and their alloys are also options for substrates ofthe array. In addition, many ceramics and polymers may also be used assubstrates. Polymers which may be used as substrates include, but arenot limited to, the following: polystyrene; poly(tetra)fluoroethylene(PTFE); polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate;polyvinylethylene; polyethyleneimine; poly(etherether)ketone;polyoxymethylene (POM); polyvinylphenol; polylactides;polymethacrylimide (PI); polyalkenesulfone (PAS); polypropylene;polyethylene; polyhydroxyethylmethacrylate (HEMA); polydimethylsiloxane;polyacrylanmide; polyimide; and block-copolymers. Preferred substratesfor the array include silicon, silica, glass, and polymers. Thesubstrate on which the patches reside may also be a combination of anyof the aforementioned substrate materials.

[0082] An array of the present invention may optionally further comprisea coating between the substrate and organic thinfilm on the array. Thiscoating may either be formed on the substrate or applied to thesubstrate. The substrate can be modified with a coating by usingthin-film technology based, for example, on physical vapor deposition(PVD), thermal processing, or plasma-enhanced chemical vapor deposition(PECVD). Alternatively, plasma exposure can be used to directly activateor alter the substrate and create a coating. For instance, plasma etchprocedures can be used to oxidize a polymeric surface (i.e., polystyreneor polyethylene to expose polar functionalities such as hydroxyls,carboxylic acids, aldehydes and the like).

[0083] The coating is optionally a metal film. Possible metal filmsinclude aluminum, chromium, titanium, tantalum, nickel, stainless steel,zinc, lead, iron, copper, magnesium,. manganese, cadmium, tungsten,cobalt, and alloys or oxides thereof. In a preferred embodiment, themetal film is a noble metal film. Noble metals that may be used for acoating include, but are not limited to, gold, platinum, silver, andcopper. In an especially preferred embodiment, the coating comprisesgold or a gold alloy. Electron-beam evaporation may be used to provide athin coating of gold on the surface of the substrate. In a preferredembodiment, the metal film is from about 50 nm to about 500 nm inthickness. In an alternative embodiment, the metal film is from about 1nm to about 1 μm in thickness.

[0084] In alternative embodiments, the coating comprises a compositionselected from the group consisting of silicon, silicon oxide, titania,tantalum oxide, silicon nitride, silicon hydride, indium tin oxide,magnesium oxide, alumina, glass, hydroxylated surfaces, and polymers.

[0085] In one embodiment of the invention array, the surface of thecoating is atomically flat. In this embodiment, the mean roughness ofthe surface of the coating is less than about 5 angstroms for areas ofat least 25 μm². In a preferred embodiment, the mean roughness of thesurface of the coating is less than about 3 angstroms for areas of atleast 25 μm². The ultraflat coating can optionally be atemplate-stripped surface as described in Hegner et al., SurfaceScience, 1993, 291:3946 and Wagner et al., Langmuir, 1995, 11:3867-3875,both of which are incorporated herein by reference.

[0086] It is contemplated that the coatings of many arrays will requirethe addition of at least one adhesion layer between said coating and thesubstrate. Typically, the adhesion layer will be at least 6 angstromsthick and may be much thicker. For instance, a layer of titanium orchromium may be desirable between a silicon wafer and a gold coating. Inan alternative embodiment, an epoxy glue such as Epo-tek 377®, Epo-tek301-2®, (Epoxy Technology Inc., Billerica, Mass.) may be preferred toaid adherence of the coating to the substrate. Determinations as to whatmaterial should be used for the adhesion layer would be obvious to oneskilled in the art once materials are chosen for both the substrate andcoating. In other embodiments, additional adhesion mediators orinterlayers may be necessary to improve the optical properties of thearray, for instance, in waveguides for detection purposes.

[0087] Deposition or formation of the coating (if present) on thesubstrate is performed prior to the formation of the organic thinfilmthereon. Several different types of coating may be combined on thesurface. The coating may cover the whole surface of the substrate oronly parts of it. The pattern of the coating may or may not be identicalto the pattern of organic thinfilms used to immobilize the proteins. Inone embodiment of the invention, the coating covers the substratesurface only at the site of the patches of the immobilized. Techniquesuseful for the formation of coated patches on the surface of thesubstrate which are organic thinfilm compatible are well known to thoseof ordinary skill in the art. For instance, the patches of coatings onthe substrate may optionally be fabricated by photolithography,micromolding (PCT Publication WO 96/29629), wet chemical or dry etching,or any combination of these.

[0088] The organic thinfilm on which each of the patches of proteins isimmobilized forms a layer either on the substrate itself or on a coatingcovering the substrate. The organic thinfilm on which the proteins ofthe patches are immobilized is preferably less than about 20 nm thick.In some embodiments of the invention, the organic thinfilm of each ofthe patches may be less than about 10 nm thick.

[0089] A variety of different organic thinfilms are suitable for use inthe present invention. Methods for the formation of organic thinfilmsinclude in situ growth from the surface, deposition by physisorption,spin-coating, chemisorption, self-assembly, or plasma-initiatedpolymeriztion from gas phase. For instance, a hydrogel composed of amaterial such as dextran can serve as a suitable organic thinfilm on thepatches of the array. In one preferred embodiment of the invention, theorganic thinfilm is a lipid bilayer. In another preferred embodiment,the organic thinfilm of each of the patches of the array is a monolayer.A monolayer of polyarginine or polylysine adsorbed on a negativelycharged substrate or coating is one option for the organic thinfilm.Another option is a disordered monolayer of tethered polymer chains. Ina particularly preferred embodiment, the organic thinfilm is aself-assembled monolayer. A monolayer of polylysine is one option forthe organic thinfilm. The organic thinfilm is most preferably aself-assembled monolayer which comprises molecules of the formula X-R-Y,wherein R is a spacer, X is a functional group that binds R to thesurface, and Y is a functional group for binding proteins onto themonolayer. In an alternative preferred embodiment, the self-assembledmonolayer is comprised of molecules of the formula (X)_(a)R(Y)_(b) wherea and b are, independently, integers greater than or equal to 1 and X R,and Y are as previously defined. In an alternative preferred embodiment,the organic thinfilm comprises a combination of organic thinfilms suchas a combination of a lipid bilayer immobilized on top of aself-assembled monolayer of molecules of the formula X-R-Y. As anotherexample, a monolayer of polylysine can also optionally be combined witha self-assembled monolayer of molecules of the formula X-R-Y (see U.S.Pat. No. 5,629,213).

[0090] In all cases, the coating, or the substrate itself if no coatingis present, must be compatible with the chemical or physical adsorptionof the organic thinfilm on its surface. For instance, if the patchescomprise a coating between the substrate and a monolayer of molecules ofthe formula X-R-Y, then it is understood that the coating must becomposed of a material for which a suitable functional group X isavailable. If no such coating is present, then it is understood that thesubstrate must be composed of a material for which a suitable functionalgroup X is available.

[0091] In a preferred embodiment of the invention, the regions of thesubstrate surface, or coating surface, which separate the patches ofproteins are free of organic thinfilm. In an alternative embodiment, theorganic thinfilm extends beyond the area of the substrate surface, orcoating surface if present, covered by the protein patches. Forinstance, optionally, the entire surface of the array may be covered byan organic thinfilm on which the plurality of spatially distinct patchesof proteins reside. An organic thinfilm which covers the entire surfaceof the array may be homogenous or may optionally comprise patches ofdiffering exposed functionalities useful in the immobilization ofpatches of different proteins. In still another alternative embodiment,the regions of the substrate surface, or coating surface if a coating ispresent, between the patches of proteins are covered by an organicthinfilm, but an organic thinfilm of a different type than that of thepatches of proteins. For instance, the surfaces between the patches ofproteins may be coated with an organic thinfilm characterized by lownon-specific binding properties for proteins and other analytes.

[0092] A variety of techniques may be used to generate patches oforganic thinfilm on the surface of the substrate or on the surface of acoating on the substrate. These techniques are well known to thoseskilled in the art and will vary depending upon the nature of theorganic thinfilm, the substrate, and the coating if present. Thetechniques will also-vary depending on the structure of the underlyingsubstrate and the pattern of any coating present on the substrate. Forinstance, patches of a coating which is highly reactive with an organicthinfilm may have already been produced on the substrate surface. Arraysof patches of organic thinfilm can optionally be created bymicrofluidics printing, microstamping (U.S. Pat. Nos. 5,512,131 and5,731,152), or microcontact printing (μCP) (PCT Publication WO96/29629). Subsequent immobilization of proteins to the reactivemonolayer patches results in two-dimensional arrays of the agents.Inkjet printer heads provide another option for patterning monolayerX-R-Y molecules, or components thereof, or other organic thinfilmcomponents to nanometer or micrometer scale sites on the surface of thesubstrate or coating (Lemmo et al., Anal Chem., 1997, 69:543-551; U.S.Pat. Nos. 5,843,767 and 5,837,860). In some cases, commerciallyavailable arrayers based on capillary dispensing (for instance,OmniGridT™ from Genemachines, inc, San Carlos, Calif., andHigh-Throughput Microarrayer from Intelligent Bio-Instruments,Cambridge, Mass.) may also be of use in directing components of organicthinfilms to spatially distinct regions of the array.

[0093] Diffusion boundaries between the patches of proteins immobilizedon organic thinfilms such as self-assembled monolayers may be integratedas topographic patterns (physical barriers) or surface functionalitieswith orthogonal wetting behavior (chemical barriers). For instance,walls of substrate material or photoresist may be used to separate someof the patches from some of the others or all of the patches from eachother. Alternatively, non-bioreactive organic thinfilms, such asmonolayers, with different wettability may be used to separate patchesfrom one another.

[0094] In a preferred embodiment of the invention, each of the patchesof proteins comprises a self-assembled monolayer of molecules of theformula X-R-Y, as previously defined, and the patches are separated fromeach other by surfaces free of the monolayer.

[0095]FIG. 1 shows the top view of one example of an array of 25 patchesreactive with proteins. On the array, a number of patches 15 cover thesurface of the substrate 3.

[0096]FIG. 2 shows a detailed cross section of a patch 15 of the arrayof FIG. 1. This view illustrates the use of a coating 5 on the substrate3. An adhesion interlayer 6 is also included in the patch. On top of thepatch resides a self-assembled monolayer 7.

[0097]FIG. 3 shows a cross section of one row of the patches 15 of thearray of FIG. 1. This figure also shows the use of a cover 2 over thearray. Use of the cover 2 creates an inlet port 16 and an outlet port 17for solutions to be passed over the array.

[0098] A variety of chemical moieties may function as monolayermolecules of the formula X-R-Y in the array of the present invention.However, three major classes of monolayer formation are preferably usedto expose high densities of reactive omegafinctionalities on the patchesof the array: (i) alkylsiloxane monolayers (“silanes”) on hydroxylatedand non-hydroxylated surfaces (as taught in, for example, U.S. Pat. No.5,405,766, PCT Publication WO 96/38726, U.S. Pat. No. 5,412,087, andU.S. Pat. No. 5,688,642); (ii) alkyl-thiol/dialkyldisulfide monolayerson noble metals (preferably Au(111)) (as, for example, described inAllara et al., U.S. Pat. No. 4,690,715; Bamdad et a., U.S. Pat. No.5,620,850; Wagner et al., Biophysical Journal, 1996, 70:2052-2066); and(iii) alkyl monolayer formation on oxide-free passivated silicon (astaught in, for example, Linford et al., J. Am. Chem. Soc., 1995,117:3145-3155, Wagner et al., Journal of Structural Biology, 1997,119:189-201, U.S. Pat. No. 5,429,708). One of ordinary skill in the art,however, will recognize that many possible moieties may be substitutedfor X, R, and/or Y, dependent primarily upon the choice of substrate,coating, and affinity tag. Many examples of monolayers are described inUlman, An Introduction to Ultrathin Organic Films: FromLangmuir-Blodgett to Self Assembly, Academic press (1991).

[0099] In one embodiment, the monolayer comprises molecules of theformula (X)_(a)R(Y)_(b) wherein a and b are, independently, equal to aninteger between 1 and about 200. In a preferred embodiment, a and b are,independently, equal to an integer between 1 and about 80. In a morepreferred embodiment, a and b are, independently, equal to 1 or 2. In amost preferred embodiment, a and b are both equal to 1 (molecules of theformula X-RY).

[0100] If the patches of the invention array comprise a self-assembledmonolayer of molecules of the formula (X)_(a)R(Y)_(b), then R mayoptionally comprise a linear or branched hydrocarbon chain from about 1to about 400 carbons long. The hydrocarbon chain may comprise an alkyl,aryl, alkenyl, alkynyl, cycloalkyl, alkaryl, aralkyl group, or anycombination thereof. If a and b are both equal to one, then R istypically an alkyl chain from about 3 to about 30 carbons long. In apreferred embodiment, if a and b are both equal to one, then R is analkyl chain from about 8 to about 22 carbons long and is, optionally, astraight alkane. However, it is also contemplated that in an alternativeembodiment, R may readily comprise a linear or branched hydrocarbonchain from about 2 to about 400 carbons long and be interrupted by atleast one hetero atom. The interrupting hetero groups can include —O—,—CONH—, —CONHCO—, —NH—, —CSNH—, —CO—, —CS—, —S—, —SO—, —(OCH₂CH₂)_(n)—(where n=1-20), —(CF₂)_(n)— (where n=1-22), and the like. Alternatively,one or more of the hydrogen moieties of R can be substituted withdeuterium. In alternative, less preferred, embodiments, R may be morethan about 400 carbons long.

[0101] X may be chosen as any group which affords chemisorption orphysisorption of the monolayer onto the surface of the substrate (or thecoating, if present). When the substrate or coating is a metal or metalalloy, X, at least prior to incorporation into the monolayer, can in oneembodiment be chosen to be an asymmetrical or symmetrical disulfide,sulfide, diselenide, selenide, thiol, isonitrile, selenol, a trivalentphosphorus compound, isothiocyanate, isocyanate, xanthanate,thiocarbamate, a phosphine, an amine, thio acid or a dithio acid. Thisembodiment is especially preferred when a coating or substrate is usedthat is a noble metal such as gold, silver, or platinum.

[0102] If the substrate of the array is a material such as silicon,silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz,glass, or silica, then the array of one embodiment of the inventioncomprises an X that, prior to incorporation into said monolayer, is amonohalosilane, dihalosilane, trihalosilane, trialkoxysilane,dialkoxysilane, or a monoalkoxysilane. Among these silanes,trichlorosilane and trialkoxysilane are particularly preferred.

[0103] In a preferred embodiment of the invention, the substrate isselected from the group consisting of silicon, silicon dioxide, indiumtin oxide, alumina, glass, and titania; and X, prior to incorporationinto said monolayer, is selected from the group consisting of amonohalosilane, dihalosilane, trihalosilane, trichlorosilane,trialkoxysilane, dialkoxysilane, monoalkoxysilane, carboxylic acids, andphosphates.

[0104] In another preferred embodiment of the invention, the substrateof the array is silicon and X is an olefin.

[0105] In still another preferred embodiment of the invention, thecoating (or the substrate if no coating is present) is titania ortantalum oxide and X is a phosphate.

[0106] In other embodiments, the surface of the substrate (or coatingthereon) is composed of a material such as titanium oxide, tantalumoxide, indium tin oxide, magnesium oxide, or alumina where X is acarboxylic acid or phosphoric acid. Alternatively, if the surface of thesubstrate (or coating thereon) of the array is copper, then X mayoptionally be a hydroxamic acid.

[0107] If the substrate used in the invention is a polymer, then in manycases a coating on the substrate such as a copper coating will beincluded in the array. An appropriate functional group X for the coatingwould then be chosen for use in the array. In an alternative embodimentcomprising a polymer substrate, the surface of the polymer may beplasma-modified to expose desirable surface functionalities formonolayer formation. For instance, EP 780423 describes the use of amonolayer molecule that has an alkene X functionality on a plasmaexposed surface. Still another possibility for the invention arraycomprised of a polymer is that the surface of the polymer on which themonolayer is formed is functionalized by copolymerization ofappropriately functionalized precursor molecules.

[0108] Another possibility is that prior to incorporation into themonolayer, X can be a free-radical-producing moiety. This functionalgroup is especially appropriate when the surface on which the monolayeris formed is a hydrogenated silicon surface. Possible free-radicalproducing moieties include, but are not limited to, diacylperoxides,peroxides, and azo compounds. Alternatively, unsaturated moieties suchas unsubstituted alkenes, alkynes, cyano compounds and isonitrilecompounds can be used for X, if the reaction with X is accompanied byultraviolet, infrared, visible, or microwave radiation.

[0109] In alternative embodiments, X, prior to incorporation into themonolayer, may be a hydroxyl, carboxyl, vinyl, sulfonyl, phosphoryl,silicon hydride, or an amino group.

[0110] The component, Y, of the monolayer is a functional groupresponsible for binding a protein onto the monolayer. In a preferredembodiment of the invention, the Y group is either highly reactive(activated) towards the protein or is easily converted into such anactivated form. In a preferred embodiment, the coupling of Y with theprotein occurs readily under normal physiological conditions notdetrimental to the activity of the protein. The functional group Y mayeither form a covalent linkage or a noncovalent linkage with the protein(or its affinity tag, if present). In a preferred embodiment, thefunctional group Y forms a covalent linkage with the protein or itsaffinity tag. It is understood that following the attachment of theprotein (with or without an affinity tag) to Y, the chemical nature of Ymay have changed. Upon attachment of the protein, Y may even have beenremoved from the organic thinfilm.

[0111] In one embodiment of the array of the present invention, Y is afunctional group that is activated in situ. Possibilities for this typeof functional group include, but are not limited to, such simplemoieties such as a hydroxyl, carboxyl, amino, aldehyde, carbonyl,methyl, methylene, alkene, alkyne, carbonate, aryliodide, or a vinylgroup. Appropriate modes of activation would be obvious to one skilledin the art. Alternatively, Y can comprise a functional group thatrequires photoactivation prior to becoming activated enough to trap theprotein.

[0112] In an especially preferred embodiment of the array of the presentinvention, Y is a complex and highly reactive functional moiety that iscompatible with monolayer formation and needs no in situ activationprior to reaction with the protein and/or affinity tag. Suchpossibilities, for Y include, but are not limited to, maleimide,N-hydroxysuccinimide (Wagner et al., Biophysical Journal, 1996,70:2052-2066), nitrilotriacetic acid (U.S. Pat. No. 5,620,850),activated hydroxyl haloacetyl, bromoacetyl, iodoacetyl, activatedcarboxyl, hydrazide, epoxy, aziridine, sulfonylchloride,trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole,imidazolecarbamate, vinylsulfone, succinimidylcarbonate, arylazide,anhydride, diazoacetate, benzophenone, isothiocyanate, isocyanate,imidoester, fluorobenzene, and biotin.

[0113]FIG. 4 shows one example of a monolayer on a substrate 3 In thisexample, substrate 3 comprises glass. The monolayer is thiolreactivebecause it bears a maleimidyl functional group Y.

[0114]FIG. 5 shows another example of a monolayer on a substrate 3 whichis silicon. In this case, however, a thinfilm gold coating 5 covers thesurface of the substrate 3. Also, in this embodiment, a titaniumadhesion interlayer 6 is used to adhere the coating 5 to the substrate3. This monolayer is aminoreactive because it bears anN-hydroxysuccinimidyl functional group Y.

[0115] In an alternative embodiment, the functional group Y of the arrayis selected from the group of simple functional moieties. Possible Yfunctional groups include, but are not limited to, —OH, —NH₂, —COOH,—COOR, —RSR, —PO₄ ⁻³, —OSO₃ ⁻², —SO₃ ⁻, —COO⁻, —SOO⁻, —CONR₂, —CN, —NR₂,and the like.

[0116] The monolayer molecules of the present invention can optionallybe assembled on the surface in. parts. In other words, the monolayerneed not necessarily be constructed by chemisorption or physisorption ofmolecules of the formula X-R-Y to the surface of the substrate (orcoating). Instead, in one embodiment, X may be chemisorbed orphysisorbed to the surface of the substrate (or coating) alone first.Then, R or even just individual components of R can be attached to Xthrough a suitable chemical reaction. Upon completion of addition of thespacer R to the X moiety already immobilized on the surface, Y can beattached to the ends of the monolayer molecule through a suitablecovalent linkage.

[0117] Not all self-assembled monolayer molecules on a given patch needbe identical to one another. Some patches may comprise mixed monolayers.For instance, the monolayer of an individual patch may optionallycomprise at least two different molecules of the formula X-R-Y, aspreviously described. This second X-R-Y molecule may optionallyimmobilize the same protein as the first. In addition, some of themonolayer molecules X-R-Y of a patch may have failed to attach anyprotein.

[0118] As another alternative embodiment of the invention, a mixed,self-assembled monolayer of an individual patch on the array maycomprise both molecules of the formula X-R-Y, as previously described,and molecules of the formula, X-R-V where R is a spacer, X is afunctional group that binds R to the surface, and V is a moiety which isbiocompatible with proteins and resistant to the non-specific binding ofproteins. For example, V may consist of a hydroxyl, saccharide, oroligo/polyethylene glycol moiety (EP Publication 780423).

[0119] In still another embodiment of the invention, the array comprisesat least one unreactive patch of organic thinfilm on the substrate orcoating surface which is devoid of any protein. For instance, theunreactive patch may optionally comprise a monolayer of molecules of theformula X-R-V, where R is a spacer, X is a functional group that binds Rto the surface, and V is a moiety resistant to the non-specific bindingof proteins. The unreactive patch may serve as a control patch or beuseful in background binding measurements.

[0120] Regardless of the nature of the monolayer molecules, in somearrays it may be desirable to provide crosslinking between molecules ofan individual patch's monolayer. In general, crosslinking confersadditional stability to the monolayer. Such methods are familiar tothose skilled in the art (for instance, see Ulman, An Introduction toUltrathin Organic Films: From Langmuir-Blodgett to Self-Assembly,Academic Press (1991)).

[0121] After completion of formation of the monolayer on the patches,the protein may be attached to the monolayer via interaction with theY-functional group. Y-functional groups which fail to react with anyproteins are preferably quenched prior to use of the array.

[0122] (d) Affinity Tags and Immobilization of the proteins.

[0123] In a preferred embodiment, the protein-immobilizing patches ofthe array further comprise an affinity tag that enhances immobilizationof the protein onto the organic thinfilm. The use of an affinity tag onthe protein of the array typically provides several advantages. Anaffinity tag can confer enhanced binding or reaction of the protein withthe functionalities on the organic thinfilm, such as Y if the organicthinfilm is a an X-R-Y monolayer as previously described. Thisenhancement effect may be either kinetic or thermodynamic. The affinitytag/thinfilm combination used in the patches of the array preferablyallows for immobilization of the proteins in a manner which does notrequire harsh reaction conditions that are adverse to protein stabilityor function. In most embodiments, immobilization to the organic thinfilmin aqueous, biological buffers is ideal.

[0124] An affinity tag also preferably offers immobilization on theorganic thinfilm that is specific to a designated site or location onthe protein (site-specific immobilization). For this to occur,attachment of the affinity tag to the protein must be site-specific.Site-specific immobilization helps ensure that the active site orbinding site of the immobilized protein, such as the antigen-bindingsite of the antibody moiety, remains accessible to ligands in solution.Another advantage of immobilization through affinity tags is that itallows for a common immobilization strategy to be used with multiple,different proteins.

[0125] The affinity tag is optionally attached directly, eithercovalently or noncovalently, to the protein. In an alternativeembodiment, however, the affinity tag is either covalently ornoncovalently attached to an adaptor which is either covalently ornoncovalendy attached to the protein.

[0126] In a preferred embodiment, the affinity tag comprises at leastone amino acid. The affinity tag may be a polypeptide comprising atleast two amino acids which is reactive with the functionalities of theorganic thinfilm. Alternatively, the affinity tag may be a single aminoacid which is reactive with the organic thinfilm. Examples of possibleamino acids which could be reactive with an organic thinfilm includecysteine, lysine, histidine, arginine, tyrosine, aspartic acid, glutamicacid, typtophan, serine, threonine, and glutamine. A polypeptide oramino acid affinity tag is preferably expressed as a fusion protein withthe immobilized protein of each patch. Amino acid affinity tags provideeither a single amino acid or a series of amino acids that can interactwith the functionality of the organic thinfilm, such as the Y-functionalgroup of the self-assembled monolayer molecules. Amino acid affinitytags can be readily introduced into recombinant proteins to facilitateoriented immobilization by covalent binding to the Y-functional group ofa monolayer or to a functional group on an alternative organic thinfilm.

[0127] The affinity tag may optionally comprise a poly(amino acid) tag.A poly(amino acid) tag is a polypeptide that comprises from about 2 toabout 100 residues of a single amino acid, optionally interrupted byresidues of other amino acids. For instance, the affinity tag maycomprise a poly-cysteine, polylysine, poly-arginine, or poly-histidine.Amino acid tags are preferably composed of two to twenty residues of asingle amino acid, such as, for example, histidines, lysines, arginines,cysteines, glutamines, tyrosines, or any combination of these. Accordingto a preferred embodiment, an amino acid tag of one to twenty aminoacids includes at least one to ten cysteines for thioether linkage; orone to ten lysines for amide linkage; or one to ten arginines forcoupling to vicinal dicarbonyl groups. One of ordinary skill in the artcan readily pair suitable affinity tags with a given functionality on anorganic thinfilm.

[0128] The position of the amino acid tag can be at an amino-, orcarboxy-terminus of the protein of a patch of the array, or anywherein-between, as long as the active site or binding site of the proteinremains in a position accessible for ligand interaction. Wherecompatible with the protein chosen, affinity tags introduced for proteinpurification are preferentially located at the C-terminus of therecombinant protein to ensure that only full-length proteins areisolated during protein purification. For instance, if intact antibodiesare used on the arrays, then the attachment point of the affinity tag onthe antibody is preferably located at a C-terminus of the effector (Fc)region of the antibody. If scFvs are used on the arrays, then theattachment point of the affinity tag is also preferably located at theC-terminus of the molecules.

[0129] Affinity tags may also contain one or more unnatural amino acids.Unnatural amino acids can be introduced using suppressor tRNAs thatrecognize stop codons (i.e., amber) (Noren et al., Science, 1989,244:182-188; Ellman et al., Methods Enzym., 1991, 202:301-336; Cload etal., Chem. Biol., 1996, 3:1033-1038). The tRNAs are chemicallyamino-acylated to contain chemically altered (“unnatural”) amino acidsfor use with specific coupling chemistries (i.e., ketone modifications,photoreactive groups).

[0130] In an alternative embodiment the affinity tag can comprise anintact protein, such as, but not limited to, glutathione S-transferase,an antibody, avidin, or streptavidin.

[0131] Other protein conjugation and immobilization techniques known inthe art may be adapted for the purpose of attaching affinity tags to theprotein. For instance, in an alternative embodiment of the array, theaffinity tag may be an organic bioconjugate which is chemically coupledto the protein of interest. Biotin or antigens may be chemically crosslinked to the protein. Alternatively, a chemical crosslinker may be usedthat attaches a simple functional moiety, such as a thiol or an amine tothe surface of a protein to be immobilized on a patch on the array.Alternatively, protein synthesis or protein ligation techniques known tothose skilled in the art may be used to attach an affinity tag to aprotein. For instance, intein-mediated protein ligation may optionallybe used to attach the affinity tag to the protein (Mathys, et al., Gene231:1-13, 1999; Evans, et al., Protein Science 7:2256-2264, 1998).

[0132] In an alternative embodiment of the invention, the organicthinfilm of each of the patches comprises, at least in part, a lipidmonolayer or bilayer, and the affinity tag comprises a membrane anchor.Optionally, the lipid monolayer or bilayer is immobilized on aself-assembled monolayer.

[0133]FIG. 6 shows a detailed cross section of a patch on one embodimentof the invention array. In this embodiment, a protein 10 is immobilizedon a monolayer 7 on a substrate 3. An affinity tag 8 connects theprotein 10 to the monolayer 7. The monolayer 7 is formed on a coating 5which is separated from the substrate 3 by an interlayer 6.

[0134] In an alternative embodiment of the invention, no affinity tag isused to immobilize the proteins onto the organic thinfilm. An amino acidor other moiety (such as a carbohydrate moiety) inherent to the proteinitself may instead be used to tether the protein to the reactive groupof the organic thinfilm. In preferred embodiments, the immobilization issite-specific with respect to the location of the site of immobilizationon the protein. For instance, the sulfhydryl group on the C-terminalregion of the heavy chain portion of a Fab′ fragment generated by pepsindigestion of an antibody, followed by selective reduction of thedisulfide between monovalent Fab′ fragments, may be used as the affinitytag. Alternatively, a carbohydrate moiety on the Fc portion of an intactantibody can be oxidized under mild conditions to an aldehyde groupsuitable for immobilizing the antibody on a monolayer via reaction witha hydrazide-activated Y group on the monolayer. Examples ofimmobilization of proteins without any affinity tag can be found inWagner et al., Biophys. J., 70:2437-2441, 1996 and the specificexamples, Examples 8-10, below.

[0135] When the proteins of at least some of the different patches onthe array are different from each other, different solutions, eachcontaining a different, preferably, affinity-tagged protein, must bedelivered to their individual patches. Solutions of proteins may betransferred to the appropriate patches via arrayers which are well-knownin the art and even commercially available. For instance,microcapillary-based dispensing systems may be used. These dispensingsystems are preferably automated and computer-aided. A description ofand building instructions for an example of a microarrayer comprising anautomated capillary system can be found on the internet athttp://cmgm.stanford.edu/pbrown/array.html andhttp://cmgm.stanford.edu/pbrown/mguidelindex.html. The use of othermicroprinting techniques for transferring solutions containing theproteins to the protein-reactive patches is also possible. Inkjetprinter heads may also optionally be used for precise delivery of theproteins to the protein-reactive patches. Representative, non-limitingdisclosures of techniques useful for depositing the proteins on thepatches may be found, for example, in U.S. Pat. No. 5,731,152 (stampingapparatus), U.S. Pat. No. 5,807,522 (capillary dispensing device), U.S.Pat. No. 5,837,860 (ink-jet printing technique, Hamilton 2200 roboticpipetting delivery system), and U.S. Pat. No. 5,843,767 (ink-jetprinting technique, Hamilton 2200 robotic pipetting delivery system),all incorporated by reference herein.

[0136] (e) Adaptors.

[0137] Another embodiment of the arrays of the present inventioncomprises an adaptor that links the affinity tag to the immobilizedprotein. The additional spacing of the protein from the surface of thesubstrate (or coating) that is afforded by the use of an adaptor isparticularly advantageous since proteins are known to be prone tosurface inactivation. The adaptor may optionally afford some additionaladvantages as well. For. instance, the adaptor may help facilitate theattachment of the protein to the affinity tag. In another embodiment,the adaptor may help facilitate the use of a particular detectiontechnique with the array. One of ordinary skill in the art will be ableto choose an adaptor which is appropriate for a given affinity tag. Forinstance, if the affinity tag is streptavidin, then the adaptor could bea biotin molecule that is chemically conjugated to the protein which isto be immobilized.

[0138] In a preferred embodiment, the adaptor is a protein. In apreferred embodiment, the affinity tag, adaptor, and protein to beimmobilized together compose a fusion protein. Such a fusion protein maybe readily expressed using standard recombinant DNA technology. Adaptorswhich are proteins arm especially useful to increase the solubility ofthe protein of interest and to increase the distance between the surfaceof the substrate or coating and the protein of interest. Use of anadaptor which is a protein can also be very useful in facilitating thepreparative steps of protein purification by affinity binding prior toimmobilization on the array. Examples of possible adaptors which areproteins include glutathione-S-transferase (GST), maltose-bindingprotein, chitin-binding protein, thioredoxin, green-fluorescent protein(GFP). GFP can alsq be used for quantification of surface binding. Ifthe protein immobilized on the patches of the array is an antibody orantibody fragment comprising an Fc region, then the adaptor mayoptionally be protein G, protein A, or recombinant protein A/G (a genefusion product secreted from a nonpathogenic form of Bacillus whichcontains four Fc binding domains from protein A and two from protein G).

[0139]FIG. 7 shows a cross section of a patch on one particularembodiment of the invention array. The patch comprises a protein 10immobilized on a monolayer 7 via both an affinity tag 8 and an adaptormolecule 9. The monolayer 7 rests on a coating 5. An interlayer 6 isused between the coating S and the substrate 3.

[0140] (f) Preparation of the Proteins of the Array.

[0141] The proteins immobilized on the array may be produced by any ofthe variety of means known to those of ordinary skill in the art.

[0142] In preparation for immobilization to the arrays of the presentinvention, the protein can optionally be expressed from recombinant DNAeither in vivo or in vitro. The cDNA of the protein to be immobilized onthe array is cloned into an expression vector (many examples of whichare commercially available) and introduced into cells of the appropriateorganism for expression. A broad range of host cells and expressionsystems may be used to produce the proteins to be immobilized on thearray. For in vivo expression of the proteins, cDNAs can be cloned intocommercial expression vectors (Qiagen, Novagen, Clontech, for example)and introduced into an appropriate organism for expression. Expressionin vivo may be done in bacteria (for example, Escherichia coli), plants(for example, Nicotiana tabacum), lower eukaryotes (for example,Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris), orhigher eukaryotes (for example, bacculovirus-infected insect cells,insect cells, mammalian cells). For in vitro expression PCR-amplifiedDNA sequences are directly used in coupled in vitrotranscription/translation systems (for instance: Escherichia coli S30lysates from T7 RNA polymerase expressing, preferably protease-deficientstrains; wheat germ lysates; reticulocyte lysates (Promega, Pharmacia,Panvera)). The choice of organism for optimal expression depends on theextent of post-translational modifications (i.e., glycosylation,lipid-modifications) desired. One of ordinary skill in the art will beable to readily choose which host cell type is most suitable for theprotein to be immobilized and application desired.

[0143] DNA sequences encoding amino acid affinity tags and adaptorprotein sequences are engineered into the expression vectors such thatthe genes of interest can be cloned in frame either 5′ or 3′ of the DNAsequence encoding the affinity tag and adaptor.

[0144] The expressed proteins are purified by affinity chromatographyusing commercially available resins.

[0145] Preferably, production of families of related proteins involvesparallel processing from cloning to protein expression and proteinpurification. cDNAs for the protein of interest will be amplified by PCRusing cDNA libraries or EST (expressed sequence tag) clones astemplates. Any of the in vitro or in vivo expression systems describedabove can then be used for expression of the proteins to be immobilizedon the array.

[0146]Escherichia coli-based protein expression is generally the methodof choice for soluble proteins that do not require extensivepost-translational modifications for activity. Extracellular orintracellular domains of membrane proteins will be fused to proteinadaptors for expression and purification.

[0147] The entire approach can be performed using 96-well assay plates.PCR reactions are carried out under standard conditions. Oligonucleotideprimers contain unique restriction sites for facile cloning into theexpression vectors. Alternatively, the TA cloning system (Clontech) canbe used. Expression vectors contain the sequences for affinity tags andthe protein adaptors. PCR products are ligated into the expressionvectors (under inducible promoters) and introduced into the appropriatecompetent Escherichia coli strain by calcium-dependent transformation(strains include: XL-1 blue, BL21, SG13009(1on-)). TransformedEscherichia coli cells are plated and individual colonies transferredinto 96-array blocks. Cultures are grown to mid-log phase, induced forexpression, and cells collected by centrifugation. Cells are resuspendedcontaiinng lysozyme and the membranes broken by rapid freeze/thawcycles, or by sonication. Cell debris is removed by centrifugation andthe supernatants transferred to 96-tube arrays. The appropriate affinitymatrix is added, protein of interest bound and nonspecifically boundproteins removed by repeated washing steps using 12-96 pin suctiondevices and centrifugation. Alternatively, magnetic affinity beads andfiltration devices can be used (Qiagen). The proteins are eluted andtransferred to a new 96-well array. Protein concentrations aredetermined and an aliquot of each protein is spotted onto anitrocellulose filter and verified by Western analysis using an antibodydirected against the affinity tag. The purity of each sample is assessedby SDS-PAGE and silver staining or mass spectrometry. Proteins aresnap-frozen and stored at −80° C.

[0148]Saccharomyces cerevisiae allows for core glycosylation and lipidmodifications of proteins. The approach described above for Escherichiacoli can be used with slight modifications for transformation and celllysis. Transformation of Saccharomyces cerevisiae is by lithium-acetateand cell lysis is either by lyticase digestion of the cell wallsfollowed by freeze-thaw, sonication or glass-bead extraction. Variationsof post-translational modifications can be obtained by different yeaststrains (i.e. Saccharomyces pombe, Pichia pastoris).

[0149] The advantage of the bacculovirus system or mammalian cells arethe wealth of post-translational modifications that can be obtained. Thebacculo-system requires cloning of viruses, obtaining high titer stocksand infection of liquid insect cell suspensions (cells are SF9, SF21).Mammalian cell-based expression requires transfection and cloning ofcell lines. Soluble proteins are collected from the medium whileintracellular or membrane bound proteins require cell lysis (eitherdetergent solubilization, freeze-thaw). Proteins can then be purifiedanalogous to the procedure described for Escherichia coli.

[0150] For in vitro translation the system of choice is Escherichia colilysates obtained from protease-deficient and T7 RNA polymeraseoverexpressing strains. Escherichia coli lysates provide efficientprotein expression (30-50 μg/ml lysate). The entire process is carriedout in 96-well arrays. Genes of interest are amplified by PCR usingoligonucleotides that contain the gene-specific sequences containing aT7 RNA polymerase promoter and binding site and a sequence encoding theaffinity tag. Alternatively, an adaptor protein can be fused to the geneof interest by PCR Amplified DNAs can be directly transcribed andtranslated in the Escherichia coli lysates without prior cloning forfast analysis. The proteins are then isolated by binding to an affinitymatrix and processed as described above.

[0151] Alternative systems which may be used include wheat germ extractsand reticulocyte extracts. In vitro synthesis of membrane proteins andor post-translationally modified proteins will require reticulocytelysates in combination with microsomes.

[0152] In one preferred embodiment of the invention, the proteinsimmobilized on the patches of the array are antibodies. Optionally, theimmobilized proteins may be monoclonal antibodies. The production ofmonoclonal antibodies against specific protein targets is routine usingstandard hybridoma technology. In fact, numerous monoclonal antibodiesare available commercially.

[0153] As an alternative to obtaining antibodies or antibody fragmentswhich have been produced by cell fusion or from continuous cell lines,the antibody moieties may be expressed in bacteriophage. Such antibodyphage display technologies are well known to those skilled in the art.The bacteriophage expression systems allow for the random recombinationof heavy- and light-chain sequences, thereby creating a library ofantibody sequences which can be selected against the desired antigen.The expression system can be based on bacteriophage λ or, morepreferably, on filamentous phage. The bacteriophage expression systemcan be used to express Fab fragments, Fv's with an engineeredintermolecular disulfide bond to stabilize the V_(H)-V_(L) pair(dsFv's), scFvs, or diabody fragments.

[0154] The antibody genes of the phage display libraries may be frompre-immunized donors. For instance, the phage display library could be adisplay library prepared from the spleens of mice previously immunizedwith a mixture of proteins (such as a lysate of human T-cells).Immunization can optionally be used to bias the library to contain agreater number of recombinant antibodies reactive towards a specific setof proteins (such as proteins found in human T-cells). Alternatively,the library antibodies may be derived from naive or synthetic libraries.The naive libraries have been constructed from spleens of mice whichhave not been contacted by external antigen. In a synthetic library,portions of the antibody sequence, typically those regions correspondingto the complementarity determining regions (CDR) loops, have beenmutagenized or randomized.

[0155] The phage display method involves batch-cloning the antibody genelibrary into a phage genome as a fusion to the gene encoding one of thephage coat proteins (pIII, pVI, or pVIII). The pIII phage protein geneis preferred. When the fusion product is expressed it is incorporatedinto the mature phage coat. As a result, the antibody is displayed as afusion on the surface of the phage and is available for binding andhence, selection, on a target protein. Once a phage particle is selectedas bearing an antibody-coat protein fusion with the desired affinitytowards the target protein, the genetic material within the phageparticle which corresponds to the displayed antibody can be amplifiedand sequenced or otherwise analyzed.

[0156] In a preferred embodiment, a phagemid is used as the expressionvector in the phage display procedures. A phagemid is a small plasmidvector that carries gene III with appropriate cloning sites and a phagepackaging signal and contains both host and phage origins ofreplication. The phagemid is unable to produce a complete phage as thegene III fusion is the only phage gene encoded on the phagemid. A viablephage can be produced by infecting cells containing the phagemid with ahelper phage containing a defective replication origin. A hybrid phageemerges which contains all of the helper phage proteins as well as thegene III-rAb fusion. The emergent phage contains the phagemid DNA only.

[0157] In a preferred embodiment of the invention, the recombinantantibodies used in phage display methods of preparing antibody fragmentsfor the arrays of the invention are expressed as genetic fusions to thebacteriophage gene III protein on a phagemid vector. For instance, theantibody variable regions encoding a single-chain Fv fragment can befused to the amino terminus of the gene III protein on a phagemid.Alternatively, the antibody fragment sequence could be fused to theamino terminus of a truncated pIII sequence lacking the first twoN-terminal domains. The phagemid DNA encoding the antibody-pIII fusionis preferably packaged into phage particles using a helper phage such asM13KO7 or VCS-M13, which supplies all structural phage proteins.

[0158] To display Fab fragments on phage, either the light or heavy (Fd)chain is fused via its C-terminus to pIII. The partner chain isexpressed without any fusion to pIII so that both chains can associateto form an intact Fab fragment. Any method of selection may be usedwhich separates those phage particles which do bind the target proteinfrom those which do not. The selection method must also allow for therecovery of the selected phages. Most typically, the phage particles areselected on an immobilized target protein. Some phage selectionstrategies known to those skilled in the art include the following:panning on an immobilized antigen; panning on an immobilized antigenusing specific elution; using biotinylated antigen and then selecting ona streptavidin resin or streptavidin-coated magnetic beads; affinitypurification; selection on Western blots (especially useful for unknownantigens or antigens difficult to purify); in vivo selection; andpathfinder selection. If the selected phage particles are amplifiedbetween selection rounds, multiple iterative rounds of selection mayoptionally be performed.

[0159] Elution techniques will vary depending upon the selection processchosen, but typical elution techniques include washing with one of thefollowing solutions: HCl or glycine buffers; basic solutions such astriethylamine; chaotropic agents; solutions of increased ionic strength;or DTT when biotin is linked to the antigen by a disulfide bridge. Othertypical methods of elution include enzymatically cleaving a proteasesite engineered between the antibody and gene III, or by competing forbinding with excess antigen or excess antibodies to the antigen.

[0160] A method for producing an array of antibody fragments thereforecomprises first selecting recombinant bacteriophage which expressantibody fragments from a phage display library. The recombinantbacteriophage are selected by affinity binding to the desired antigen.(Iterative rounds of selection are possible, but optional.) Next atleast one purified sample of an antibody fragment from a bacteriophagewhich was selected in the first step is produced. This antibodyproduction step typically entails infecting E. coli cells with theselected bacteriophage. In the absence of helper phage, the selectedbacteriophage then replicate as expressive plasmids without producingphage progeny. Alternatively, the antibody fragment gene of the selectedrecombinant bacteriophage is isolated, amplified, and then expressed ina suitable expression system. In either case, following. amplification,the expressed antibody fragment of the selected and amplifiedrecombinant bacteriophage is isolated and purified. In a third step ofthe method, the earlier steps of phage display selection and purifiedantibody fragment production are repeated using affinity binding toantigens from before until the desired plurality of purified samples ofdifferent antibody fragments with different binding partners areproduced. In a final step of the method, the antibody fragment of eachdifferent purified sample is immobilized onto organic thinfilm on aseparate patch on the surface of a substrate to form a plurality ofpatches of antibody fragments on discrete, known regions of thesubstrate surface covered by organic thinfilm.

[0161] For instance, to generate an antibody array with antibodyfragments against known antigens, open reading frames of the knownprotein targets identified in DNA databases are amplified by polymerasechain reaction and transcribed and translated in vitro to produceproteins on which a recombinant bacteriophage expressing single-chainantibody. fragments are selected. Once selected, the antibody fragmentsequence of the selected bacteriophage is amplified (typically using thepolymerase chain method) and recloned into a desirable expressionsystem. The expressed antibody fragments are purified and then printedonto organic thinfilms on substrates to form the high density arrays.

[0162] In the preparation of the arrays of the invention, phage displaymethods analogous to those used for antibody fragments may be used forother proteins which are to be immobilized on an array of the inventionas long as the protein is of suitable size to be incorporated into thephagemid or alternative vector and expressed as a fusion with abacteriophage coat protein. Phage display techniques using non-antibodylibraries typically make use of some type of protein host scaffoldstructure which supports the variable regions. For instance, β-sheetproteins, a-helical handle proteins, and other highly constrainedprotein structures have been used as host scaffolds.

[0163] Alternative display vectors may also be used to produce theproteins which are printed on the arrays of the invention. Polysomes,stable protein-ribosome-mRNA complexes, can be used to replace livebacteriophage as the display vehicle for recombinant antibody fragmentsor other proteins (Hanes and Pluckthun, Proc. Natl. Acad. Sci USA,94:4937-4942, 1997). The polysomes are formed by preventing release ofnewly synthesized and correctly folded protein from the ribosome.Selection of the polysome library is based on binding of the antibodyfragments or other proteins which are displayed on the polysomes to thetarget protein. mRNA which encodes the displayed protein or antibodyhaving the desired affinity for the target is then isolated. Largerlibraries may be used with polysome display than with phage display.

[0164] (g) Uses of the Arrays.

[0165] The present invention also provides for methods of using theinvention array. The arrays of the present invention are particularlysuited for the use in drug development. Other uses include medicaldiagnostics, proteomics and biosensors.

[0166] Use of one of the protein arrays of the present invention mayoptionally involve placing the two-dimensional protein array in aflowchamber with approximately 1-10 microliters of fluid volume per 25overall surface area. The cover over the array in the flowchamber ispreferably transparent or translucent. In one embodiment, the cover maycomprise Pyrex or quartz glass. In other embodiments, the cover may bepart of a detection system that monitors interaction between biologicalmoieties immobilized on the array and an analyte. The flowchambersshould remain filled with appropriate aqueous solutions to preserveprotein activity. Salt, temperature, and other conditions are preferablykept similar to those of normal physiological conditions. Analytes andpotential drug compounds may be flushed into the flow chamber as desiredand their interaction with the immobilized proteins determined.Sufficient time must be given to allow for binding between theimmobilized proteins and an analyte to occur. No specializedmicrofluidic pumps, valves, or mixing techniques are required for fluiddelivery to the array.

[0167] Alternatively, fluid can be delivered to each of the patches ofthe array individually. For instance, in one embodiment, the regions ofthe substrate surface may be microfabricated in such a way as to allowintegration of the array with a number of fluid delivery channelsoriented perpendicular to the array surface, each one of the deliverychannels terminating at the site of an individual protein-coated patch.

[0168] The sample which is delivered to the array is typically a fluid.

[0169] In general, delivery of solutions containing proteins to be boundby the proteins of the array may optionally be preceded, followed, oraccompanied by delivery of a blocking solution. A blocking solutioncontains protein or another moiety which will adhere to sites ofnon-specific binding on the array. For instance, solutions of bovineserum albumin or milk may be used as blocking solutions.

[0170] A wide range of detection methods is applicable to the methods ofthe invention. As desired, detection may be either quantitative orqualitative. The invention array can be interfaced with opticaldetection methods such as absorption in the visible or infrared range,chemoluminescence, and fluorescence (including lifetime, polarization,fluorescence correlation spectroscopy (FCS), and fluorescence-resonanceenergy transfer (FRET)). Furthermore, other modes of detection such asthose based on optical waveguides (PCT Publication WO 96/26432 and U.S.Pat. No. 5,677,196), surface plasmon resonance, surface charge sensors,and surface force sensors are compatible with many embodiments of theinvention. Alternatively, technologies such as those based on Brewsterangle microscopy (Schaaf et al., Langmuir, 3:1131-1135 (1987)) andellipsometry (U.S. Pat. Nos. 5,141,311 and 5,116,121; Kim,Macromolecules, 22:2682-2685 (1984)) can be used in conjunction with thearrays of the invention. Quartz crystal microbalances and desorptionprocesses (see for example, U.S. Pat. No. 5,719,060) provide still otheralternative detection means suitable for at least some embodiments ofthe invention array. An example of an optical biosensor systemcompatible both with some arrays of the present invention and a varietyof non-label detection principles including surface plasmon resonance,total internal reflection fluorescence (TIRF), Brewster Anglemicroscopy, optical waveguide lightmode spectroscopy (OWLS), surfacecharge measurements, and ellipsometry can be found in U.S. Pat. No.5,313,264.

[0171] Although non-label detection methods are generally preferred,some of the types of detection methods commonly used for traditionalimmunoassays which require the use of labels may be applied to use withat least some of the arrays of the present invention, especially thosearrays which are arrays of protein-capture agents. These techniquesinclude noncompetitive immunoassays, competitive immunoassays, and duallabel, ratiometric immunoassays. These particular techniques areprimarily suitable for use with the arrays of proteins when the numberof different proteins with different specificity is small (less thanabout 100). In the competitive method, binding-site occupancy isdetermined indirectly. In this method, the proteins of the array areexposed to a labeled developing agent, which is typically a labeledversion of the analyte or an analyte analog. The developing agentcompetes for the binding sites on the protein with the analyte. Thefractional occupancy of the proteins on different patches can bedetermined by the binding of the developing agent to the proteins of theindividual patches. In the noncompetitive method, binding site occupancyis determined directly. In this method, the patches of the array areexposed to a labeled developing agent capable of binding to either thebound analyte or the occupied binding sites on the protein. Forinstance, the developing agent may be a labeled antibody directedagainst occupied sites (i. e., a “sandwich assay”). Alternatively, adual label, ratiometric, approach may be taken where the immobilizedprotein is labeled with one label and the second, developing agent islabeled with a second label (Ekins, et al., Clinica Chimica Acta.,194:91-114, 1990). Many different labeling methods may be used in theaforementioned techniques, including radioisotopic, enzymatic,chemiluminescent, and fluorescent methods. Fluorescent methods arepreferred.

[0172]FIG. 8 shows a schematic diagram of one type of fluorescencedetection unit which may be used to monitor interaction of immobilizedproteins of an array with an analyte. In the illustrated detection unit,the protein array 21 is positioned on a base plate 20. Light from a 100Wmercury arc lamp 25 is directed through an excitation filter 24 and ontoa beam splitter 23. The light is then directed through a lens 22, suchas a Micro Nikkor 55 mm 1:2:8 lens, and onto the array 21. Fluorescenceemission from the array returns through the lens 22 and the beamsplitter 23. After next passing through an emission filter 26, theemission is received by a cooled CCD camera 27, such as the SlowscanTE/CCD-1024SF&SB (Princeton Instruments). The camera is operablyconnected to a CPU 28 which is in turn operably connected to a VCR 29and a monitor 30.

[0173]FIG. 9 shows a schematic diagram of an alternative detectionmethod based on ellipsometry. Ellipsometry allows for information aboutthe sample to be determined from the observed change in the polarizationstate of a reflected light wave. Interaction of an analyte with a layerof immobilized proteins on a patch results in a thickness change andalters the polarization status of a plane-polarized light beam reflectedoff the surface. This process can be monitored in situ from aqueousphase and, if desired, in imaging mode. In a typical setup,monochromatic light (e.g. from a He—Ne laser, 30) is plane polarized(polarizer 31) and directed onto the surface of the sample and detectedby a detector 35. A compensator 32 changes the elliptically polarizedreflected beam to plane-polarized. The corresponding angle is determinedby an analyzer 33 and then translated into the ellipsometric parametersPsi and Delta which change upon binding of analyte with the immobilizedproteins. Additional information can be found in Azzam, et al.,Ellipsometry and Polarized Light, North-Holland Publishing Company:Amsterdam, 1977.

[0174] In one embodiment, the invention provides a method for screeninga plurality of proteins for their ability to interact with a componentof a sample comprising the steps of delivering the sample to a proteinarray of the invention comprising the proteins to be screened anddetecting for the interaction of the component with the immobilizedprotein of each patch. Optionally, the component may be a protein.

[0175] Possible interactions towards which the present invention may bedirected include, but are not limited to, antibody/antigen,antibody/hapten, enzyme/substrate, carrier protein/substrate,lectin/carbohydrate, receptor/hormone, receptor/effector, protein/DNA,protein/RNA, repressor/inducer, or the like. The interaction may involvebinding and/or catalysis. The array of he invention is even suitable forassaying translocation by a membrane through a lipid bilayer. Inpreferred embodiments of use of the array, the assayed interaction is abinding interaction. The assayed interaction may be between a potentialdrug candidate and a plurality of potential drug targets. For instance,a synthesized organic compound may be tested for its ability to act asan inhibitor to a family of immobilized receptors.

[0176] Another aspect of the invention provides for a method forscreening a plurality of proteins for their ability to bind a particularcomponent of a sample. This method comprises delivering the sample to aprotein array of the invention comprising the proteins to be screenedand detecting, either directly or indirectly, for the presence or amountof the particular component retained at each patch. In a preferredembodiment, the method further comprises the intermediate step ofwashing the array to remove any unbound or nonspecifically boundcomponents of the sample from the array before the detection step. Inanother embodiment, the method further comprises the additional step offurther characterizing the particular component retained on at least onepatch. The particular component may optionally be a protein.

[0177] The optional step of further characterizing the particularcomponent retained on a patch of the array is typically designed toidentify the nature of the component bound to the protein of aparticular patch. In some cases, the entire identity of the componentmay not be known and the purpose of the further characterization may bethe initial identification of the mass, sequence, structure and/oractivity (if any) of the bound component. In other cases, the basicidentity of the component may be known, but some information about thecomponent may not be known. For instance it may be known that thecomponent is a particular protein, but the post-translationalmodification, activation state, or some other feature of the protein maynot be known. In one embodiment, the step of further characterizingcomponents which are proteins involves measuring the activity of theproteins. Although in some cases it may be preferable to remove thecomponent from the patch before the step of further characterizing theprotein is carried out, in other cases the component can be furthercharacterized while still bound to the patch. In still furtherembodiments, the proteins of the patch which binds a component can beused to isolate and/or purify the component on a larger scale, such asby purifying a component which is a protein from cells. The purifiedsample of the component can then be characterized through traditionalmeans such as microsequencing, mass spectrometry, and the like.

[0178] In another embodiment of the invention, a method of assaying forprotein-protein binding interactions is provided which comprises thefollowing steps: first, delivering a sample comprising at least oneprotein to be assayed for binding to the protein array of the invention;and then detecting, either directly, or indirectly, for the presence oramount of the protein from the sample which is retained at each patch.In a preferred embodiment, the method further comprises an additionalstep prior to the detection step which comprises washing the array toremove unbound or nonspecifically bound components of the sample fromthe array. Typically, the protein being assayed for binding will be fromthe same organism as the proteins immobilized on the array.

[0179] Another embodiment of the invention provides a method of assayingin parallel for the presence of a plurality of analytes in a samplewhich can react with one or more of the immobilized proteins on theprotein array. This method comprises delivering the sample to theinvention array and detecting for the interaction of the analyte withthe immobilized protein at each patch.

[0180] In still another embodiment of the invention, a method ofassaying in parallel for the presence of a plurality of analytes in asample which can bind one or more of the immobilized proteins on theprotein array comprises delivering the fluid sample to the inventionarray and detecting, either directly or indirectly, for the presence oramount of analyte retained at each patch. In a preferred embodiment, themethod further comprises the step of washing the array tot remove anyunbound or non-specifically bound components of the sample from thearray.

[0181] The array may be used in a diagnostic manner when the pluralityof analytes being assayed are indicative of a disease condition or thepresence of a pathogen in an organism. In such embodiments, the samplewhich is delivered to the array will then typically be derived from abody fluid or a cellular extract from the organism.

[0182] The array may be used for drug screening when a potential drugcandidate is screened directly for its ability to bind or otherwiseinteract with a plurality of proteins on. the invention array.Alternatively, a plurality of potential drug candidates may be screenedin parallel for their ability to bind or otherwise interact with one ormore immobilized proteins on the array. The drug screening process mayoptionally involve assaying for the interaction, such as binding, of atleast one analyte or component of a sample with one or more immobilizedproteins on an invention array, both in the presence and absence of thepotential drug candidate. This allows for the potential drug candidateto be tested for its ability to act as an inhibitor of the interactionor interactions originally being assayed.

[0183] (h) EXAMPLES

[0184] The following specific examples are intended to illustrate theinvention and should not be construed as limiting the scope of theclaims:

Example 1 Fabrication of a Two-Dimensional Array by Photolithography

[0185] In a preferred embodiment of the invention, two-dimensionalarrays are fabricated onto the substrate material via standardphotolithography and/or thin film deposition. Alternative techniquesinclude microcontact printing. Usually, a computer-aided design patternis transferred to a photomask using standard techniques, which is thenused to transfer the pattern onto a silicon wafer coated withphotoresist.

[0186] In a typical example, the array (“chip”) with lateral dimensionsof 10×10 mm comprises squared patches of a bioreactive layer (here: goldas the coating on a silicon substrate) each 0.1×0.1 mm in size andseparated by hydrophobic surface areas with a 0.2 mm spacing. 4″diameter Si(100) wafers (Virginia Semiconductor) are used as bulkmaterials. Si(100) wafers are first cleaned in a 3:1 mixture of H₂SO₄,conc.: 30% H₂O₂ (90° C., 10 min), rinsed with deionized water (18 MΩm),finally passivated in 1% aqueous HF, and singed at 150° C. for 30 min tobecome hydrophobic. The wafer is then spincoated with photoresist(Shipley 1813), prebaked for 25 minutes at 90° C., exposed using a KarlSuss contact printer and developed according to standard protocols. Thewafer is then dried and postbaked at 110° C. for 25 min. In the nextstep, the wafer is primed with a titanium layer of 20 nm thickness,followed by a 200 nm thick gold layer. Both layers were deposited usingelectron-beam evaporation (5 Å/s). After resist stripping and a shortplasma treatment, the gold patches can be further chemically modified toachieve the desired bioreactive and biocompatible properties (seeExample 3, below).

Example 2 Fabrication of a Two-Dimensional Array by Deposition Through aHole Mask

[0187] In another preferred embodiment the array of gold patches isfabricated by thin film deposition through a hole mask which is indirect contact with the substrate. In a typical example, Si(100) wafersare first cleaned in a 3:1 mixture of H₂SO₄, conc.: 30% H₂O₂ (90° C., 10min), rinsed with deionized water (18 MΩcm), finally passivated in 1%aqueous HF and singed at 150° C. for 30 min to become hydrophobic. Thewafer is then brought into contact with a hole mask exhibiting thepositive pattern of the desired patch array. In the next step, the waferis primed with a titanium layer of 20 nm thickness, followed by a 200 nmthick gold layer. Both layers were deposited using electron-beamevaporation (5 Å/s). After removal of the mask, the gold patches can befurther chemically modified to achieve the desired bioreactive andbiocompatible properties (see Example 3, below).

Example 3 Synthesis of an Aminoreactive Monolayer Molecule (Followingthe Procedure Outlined in Wagner et al., Biophys. J., 1996,70:2052-2066)

[0188] General.

[0189]¹H- and 13C-NMR spectra are recorded on Bruker instruments (100 to400 MHz). Chemical shifts (δ) are reported in ppm relative to internalstandard ((CH₃)₄Si, δ=0.00 (¹H- and ¹³C-NMR)). FAB-mass spectra arerecorded on a VG-SABSEQ instrument (Cs⁺, 20 keV). Transmission infraredspectra are obtained as dispersions in KBr on an FTIR Perkin-Elmer 1600Series instrument. Thin-layer chromatography (TLC) is performed onprecoated silica gel 60 F254 plates (MERCK, Darmstadt, FRG), anddetection was done using Cl₂/toluidine, PdCl₂ and UV-detection underNH3-vapor. Medium pressure liquid chromatography (MPLC) is performed ona Labomatic MD-80 (LABOMATIC INSTR. AG, Allschwil, Switzerland) using aBuechi column (460×36 mm; BUECHK FlawiL Switzerland), filled with silicagel 60 (particle size 15-40 μm) from Merck.

[0190] Synthesis of 11,11′-dithiobis(succinimidylundecanoate) (DSU).

[0191] Sodium thiosulfate (55.3 g, 350 mmol) is added to a suspension of11-bromo-undecanoic acid (92.8 g, 350 mmol) in 50% aqueous 1,4-dioxane(1000 ml). The mixture is heated at reflux (90 ° C.) for 2 h until thereaction to the intermediate Bunte salt was complete (clear solution).The oxidation to the corresponding disulfide is carried out in situ byadding iodine in portions until the solution retained with a yellow tobrown colour. The surplus of iodine is retitrated with 15% sodiumpyrosulfite in water. After removal of 1,4-dioxane by rotary evaporationthe creamy suspension is filtered to yield product11,11′-dithiobis(undecanoic acid). Recrystallization from ethylacetate/THF provides a white solid (73.4 g, 96.5%): mp 94° C.; ¹H NMR(400 MHz, CDC₃/CD₃OD 95:5): δ2.69 (t, 2H, J=7.3 Hz), 2.29 (t, 2H, J=7.5Hz), 1.76-1.57 (m, 4H), and 1.40-1.29 (m, 12H); FAB-MS (Cs⁺, 20 keV):m/z (relative intensity) 434 (100, M⁺). Anal. Calcd. for C₂₂H₄₂O₄S₂: C,60.79; H, 9.74; S, 14.75. Found: C, 60.95; H, 9.82; S, 14.74. To asolution of 11,11′-dithiobis(undecanoic acid). (1.0 g, 2.3 mmol) in THF(50 ml) is added N-hydroxysuccinimide (0.575 g, 5 mmol) followed by DCC(1.03 g, 5 mmol) at 0° C. After the reaction mixture is allowed to warmto 23° C. and is stirred for 36 h at room temperature, thedicyclohexylurea (DCU) is filtered. Removal of the solvent under reducedpressure and recrystallization from acetone/hexane provides11,11′-dithiobis(succinimidylundecanoate) as a white solid. Finalpurification is achieved by medium pressure liquid chromatography (9bar) using silica gel and a 2:1 mixture of ethyl acetate and hexane. Theorganic phase is concentrated and dried in vacuum to afford11,11′-dithiobis(succinimidylundecanoate) (1.12 g, 78%): mp 95° C.; IHNMR (400 MHz, CDCl₃): δ 2.83 (s, 4H), 2.68 (t, 2H, J=7.3 Hz), 2.60 (t,2H, J=7.5 Hz), 1.78-1.63 (m, 4H), and 1.43-1.29 (m, 12H); FAB-MS (Cs⁺,20 keV): m/z (relative intensity) 514 (100), 628 (86, M⁺). Anal. Calcd.for C₃₀H₄₈N₂O₈S₂: C, 57.30; H, 7.69; N, 4.45; S, 10.20. Found: C, 57.32;H, 7.60; N, 4.39; S, 10.25.

Example 4 Formation of an Aminoreactive Monolayer on Gold (Following theProcedure of Wagner et al., Biophys. J., 1996, 70:2052-2066)

[0192] Monolayers based on 11,11′-dithiobis(succinimidylundecanoate)(DSU) can be deposited on Au(111) surfaces of microarrays describedunder Examples 1 and 2 by immersing them into a 1 mM solution of DSU inchloroform at room temperature for 1 hour. After rinsing with 10 volumesof solvent, the N-hydroxysuccinimidyl-terminated monolayer is driedunder a stream of nitrogen and immediately used for proteinimmobilization.

Example 5 Expression and Purification of Human Caspase Fusion Proteins

[0193] Caspases are cysteine proteases of the papain superfamily, with adifferent active site and catalytic mechanism than observed for papain,Wilson, K. P. et al., Nature, 1994 370:270-275. Caspases are importantenzymes in the promotion of the cell death pathways and inflammation,Villa, et al, TIBS, 1997, 22:288-392. Identification of selectivecaspase inhibitors is essential to prevent cross-inhibition of othercaspase-dependent pathways. Caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,Villa, et al., TIBS, 1997, 22:288-392 and new caspase homologsidentified by the human genome project are PCR amplified and cloned intoan E. coli expression vector containing an N-terminal histidine tag,Hochuli, et al., Biotechnology, 1988 6:1321, a factor Xa cleavage site,a lysine tag and a tri-glycine linker. Fusion proteins are expressed,purified by nickel-nitrilotriacetic acid (NTA) agarose chromatography,the histidine tag removed by factor Xa cleavage, followed by gelfiltration. Caspases are snap-frozen and stored in 20 mM PIPES, pH 7.2,150 mM NaCl, 0.1% CHAPS, 10% sucrose at −80° C.

Example 6 Immobilization of Fusion Proteins on a 2D-Protein Array

[0194] Caspase-fusion proteins can be immobilized to the aminoreactivemonolayer surface of the bioreactive patches of the two-dimensionalarray (see Examples 1, 2, and 4 above). Caspase fusion proteins can bediluted to concentrations of 1 μg/ml in 20 mM PIPES, pH 7.2, 150 mMNaCl, 0.1% CHAPS, 10% sucrose and applied onto the bioreactive patchesusing a computer-aided, capillary-based dispensing system. After animmobilization period of 30 min, the 2D array was rinsed and subjectedto analysis. Ultrapure water with a resistance of 18 MΩm is generallyuseable for all aqueous buffers (purified by passage through a BarnsteadNanopure® system).

Example 7 Assay of Caspase Activity on a Two-Dimensional Array

[0195] Caspase activity can be determined by a binding assay using threefluorescently labeled peptide aldehyde inhibitors that form a reversiblethiohemiacetal moiety with the active site cysteine, Thornberry, Methodsin Enzymology, 1994, 244:615-631. The peptides are adapted to caspase 1,3, 4, 7: Dns (dansyl)-SS-DEVD-CHO, caspase 1: Dns-SS-VDVAD-CHO, caspase6: Dns-SS-VQID-CHO, Talanian, J. Biol. Chem., 1997, 272:9677-9682. Theaffinity for Ac-DEVD-CHO to caspase 1 is determined to be in the lownanomolar range, Thornberry, Methods in Enzymology, 1994, 244:615-631.The assay buffer is 20 mM PIPES, pH 7.2, 150 mM NaCl, 0.1% CHAPS, 10%sucrose, Stennicke, and Salvesen, J. Biol. Chem., 1997, 272:25719-25723.Fluorescently labeled peptides are mixed to a final concentration of 1to 5 nM each, the potential drug compound added and flushed onto the 2Darray. Peptides are allowed to bind for 10-60 min., unbound peptideremoved by washing with buffer and the fluorescence intensity measured(excitation at 360 nm, emission at 470 nm).

Example 8 Formation and Use of an Array of Immobilized Fab′ AntibodyFragments to Detect Concentrations of Soluble Proteins Prepared fromCultured Mammalian Cells

[0196] Collections of IgG antibodies are purchased from commercialsources (e.g. Pierce, Rockford, Ill.). The antibodies are first purifiedby affinity chromatography based on binding to immobilized protein A.The antibodies are diluted 1:1 in binding buffer(0.1 M Tris-HCl, 0.15 MNaCl, pH 7.5). A 2 ml minicolumn containing a gel with immobilizedprotein A is prepared. (Hermanson, et. al., Immobilized Affinity LigandTechniques, Academic Press, San Diego, 1992.) The column is equilibratedwith 10 ml of binding buffer. Less than 10 mg of immunoglobulin isapplied to each 2 ml minicolumn and the column is washed with bindingbuffer until the absorbance at 280 nm is less than 0.02. The boundimmunoglobulins are eluted with 0.1 M glycine, 0.15 M NaCl, pH 2.8, andimmediately neutralized with 1.0 M Tris-HCl, pH 8.0 to 50 mM finalconcentration and then dialyzed against 10 mM sodium phosphate, 0.15 MNaCl, pH 7.2 and stored at 4° C.

[0197] The purified immunoglobulin are digested with immobilized pepsin.Pepsin is an acidic endopeptidase and hydrolyzes proteins favorablyadjacent to aromatic and dicarboxylic L-amino acid residues. Digestionof IgG with pepsin generates intact F(ab′) fragments. Immobilized pepsingel is washed with digestion buffer; 20 mM sodium acetate, pH 4.5. Asolution of purified IgG at 10 mg/ml is added to the immobilized pepsingel and incubated at 37° C. for 2 hours. The reaction is neutralized bythe addition of 10 mM Tris-HCl, pH 7.5 and centrifuged to pellet thegel. The supernatant liquid is collected and applied to an immobilizedprotein A column, as described above, to separate the F(ab′) ₂ fragmentsfrom the Fc and undigested IgG. The pooled F(ab′)₂ is dialyzed against10 mM sodium phosphate, 0.15 M NaCl, pH 7.2 and stored at 4° C. Thequantity of pooled, eluted F(ab′)₂ is measured by peak area absorbanceat 280 nm.

[0198] The purified F(ab′)₂ fragments at a concentration of 10 mg/ml arereduced at 37° C. for 1 hour in a buffer of 10 mM sodium phosphate, 0.15M NaCl, 10 mM 2mercaptoethylamine, 5 mM EDTA, pH 6.0. The Fab′ fragmentsare separated from unsplit F(ab′)₂ fragments and concentrated byapplication to a Sephadex G-25 column (M_(r)=46,000-58,000). The pooledFab′ fragments are dialyzed against 10 mM sodium phosphate, 0.15 M NaCl,pH 7.2. The reduced Fab′ fragments are diluted to 100 μg/ml s andapplied onto the bioreactive patches containing exposed aminoreactivefunctional groups using a computer-aided, capillary-basedmicrodispensing system (for antibody immobilization procedures, seeDammer et al., Biophys. J, 70:2437-2441, 1996). After an immobilizationperiod of 30 minutes at 30° C., the array is rinsed extensively with 10mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.

[0199] Transformed human cells grown in culture are collected by lowspeed centrifugation, briefly washed with ice-cold phosphate-bufferedsolution (PBS), and then resuspended in ice-cold hypotonic buffercontaining DNase/RNase (10 μg/ml each, final concentration) and amixture of protease inhibitors. Cells are transferred to amicrocentrifuge tube, allowed to swell for 5 minutes, and lysed by rapidfreezing in liquid nitrogen and thawing in ice-cold water. Cell debrisand precipitates are removed by high-speed centrifugation and thesupernatant is cleared by passage through a 0.45 μM filter. The clearedlysate is applied to the Fab′ fragment array described above and allowedto incubate for 2 hours at 30° C. After binding the array is washedextensively with 10 mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.The location and amount of bound proteins are determined by opticaldetection.

Example 9 Formation and Use of an Array of Immobilized AntibodyFragments to Detect Concentrations of Soluble Proteins Prepared fromCultured Mammalian Cells

[0200] A combinatorial library of filamentous phage expressing scFvantibody fragments is generated based on the technique of McCafferty andcoworkers; McCafferty, et al., Nature, 1990, 348:552-554; Winter andMilstein, Nature, 1991, 349:293-299. Briefly, mRNA is purified frommouse spleens and used to construct a cDNA library. PCR fragmentsencoding sequences of the variable heavy and light chain immunoglobulingenes of the mouse are amplified from the prepared cDNA. The amplifiedPCR products are joined by a linker region of DNA encoding the 15 aminoacid peptide (Gly₄SerGly₂CysGlySerGly₄Ser) (SEQ ID NO: 1) and theresulting fill-length PCR fragment is cloned into an expression plasmid(PCANTAB 5 E) in which the purification peptide tag (E Tag) has beenreplaced by a His₆ peptide (SEQ ID NO: 2). Electrocompetent TG1 E. colicells are transformed with the expression plasmid by electroporation.The pCANTAB-transformed cells are induced to produced functionalfilamentous phage expressing scFv fragments by superinfection withM13KO7 helper phage. Cells are grown on glucose-deficient mediumcontaining the antibiotics ampicillin (to select for cells with thephagemid) and kanamycin (to select for cells infected with M13KO7). Inthe absence of glucose, the lac promoter present on the phagemid is nolonger repressed, and synthesis of the scFv-gene 3 fusion begins.

[0201] Proteins from a cell lysate are adsorbed to the wells of a96-well plate. Transformed human cells grown in culture are collected bylow speed centrifugation and the cells are briefly washed with ice-coldPBS. The washed cells are then resuspended in ice-cold hypotonic buffercontaining DNase/RNase (10 μg/ml each, final concentration) and amixture of protease inhibitors, allowed to swell for 5 minutes, andlysed by rapid freezing in liquid nitrogen and thawing in ice-coldwater. Cell debris and precipitates are removed by high-speedcentrifugation and the supernatant is cleared by passage through a 0.45μm filter. The cleared lysate is diluted to 10 μg/ml in dilution buffer;20 mM PIPES, 0.15 M NaCl, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM2-mercaptoethanol, 2 mM DTT, pH 7.2 and applied to the 96-plate wells.After immobilization for 1 hour at 30° C., the well is washed with thedilution buffer and then incubated with dilution buffer containing 10%nonfat dry milk to block unreacted sites. After the blocking step, thewell is washed extensively with the dilution buffer.

[0202] Phage expressing displayed antibodies are separated from E. colicells by centrifugation and then precipitated from the supernatant bythe addition of 15% w/v PEG 8000, 2.5 M NaCl followed by centrifugation.The purified phage are resuspended in the dilution buffer containing 3%nonfat dry milk and applied to the well containing the immobilizedproteins described above, and allowed to bind for 2 hours at 37° C.,followed by extensive washing with the binding buffer. Phage are elutedfrom the well with an elution buffer; 20 mM PIPES, 1 M NaCl, 0.1% CHAPS,10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. The well isthen extensively washed with purge buffer; 20 mM PIPES, 2.5 M NaCl, 0.1%CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. Thewell is then extensively washed with dilution buffer; 20 mM PIPES, 0.15M NaCl, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH7.2. The eluted phage solution is then re-applied to a new wellcontaining adsorbed antigen and the panning enrichment is repeated 4times. Finally, the phage are eluted from the well with 2M of NaCl in 20mM PIPES, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT,pH 7.2. Eluates are collected and mixed with log-phase TG1 cells, andgrown at 37° C. for 1 hour and then plated onto SOB medium containingampicillin and glucose and allowed to grow for 12-24 hours.

[0203] Individual colonies are picked and arrayed into 96-well 2mlblocks containing SOB medium and M13KO7 helper phage and grown for 8hours with shaking at 37° C. The phage are separated from cells bycentrifugation and precipitated with PEG/NaCl as described above.Concentrated phage are used to infect HB2151 E. coli. E. coli TG1produces a suppressor tRNA which allows readthrough (suppression) of anamber stop codon located between the scFv and phage gene 3 sequences ofthe pCANTAB 5 E plasmid. Infected HB2151 cells are selected on mediumcontaining ampicillin, glucose, and nalidixic acid. Cells are grown tomid-log and then centrifiged and resuspended in medium lacking glucoseand growth continued Soluble scFv fragments will accumulate in the cellperiplasm. A periplasmic extract is prepared from pelleted cells by mildosmotic shock. The soluble scFv released into the supernatant ispurified by affinity binding to Ni-NTA activated agarose and eluted with10 mM EDTA.

[0204] The purified scFv antibody fragments are diluted to 100 μg/ml andapplied onto the bioreactive patches with exposed aminoreactive groupsusing a computer-aided, capillary-based microdispensing system. After animmobilization period of 30 minutes at 30° C., the array is rinsedextensively with 10 mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.

[0205] Transformed human cells grown in culture are collected by lowspeed centrifugation, briefly washed with ice-cold PBS, and thenresuspended in ice-cold hypotonic buffer containing DNase/RNase (10μg/ml each, final concentration) and mixture of protease inhibitors.Cells are transferred to a microcentrifige tube, allowed to swell for 5minutes, and lysed by rapid freezing in liquid nitrogen and thawing inice-cold water. Cell debris and precipitates are removed by high-speedcentrifugation and the supernatant is cleared by passage through a 0.45μm filter. The cleared lysate is applied to the scFv fragment arraydescribed above and allowed to incubate for 2 hours at 30° C. Afterbinding, the array is washed extensively with 0.1 M sodium phosphate,0.15 M NaCl, 5 mM EDTA pH 7.0. The location and amount of bound proteinsare determined by optical detection.

[0206] Patterns of binding are established empirically by testingdilutions of a control ,cell extract. Extracts from experimental cellsare diluted to a series of concentrations and then tested against thearray. Patterns of protein expression in the experimental cell lysatesare compared to protein expression patterns in the control samples toidentify proteins with unique expression profiles.

Example 10 Formation and Use of an Array of Immobilized MonoclonalAntibodies to Detect Concentrations of Soluble Proteins Prepared fromCultured Mammalian Cells

[0207] Collections of monoclonal antibodies are purchased fromcommercial suppliers as either raw ascities fluid or purified bychromotography over protein A, protein G, or protein L. If from rawascites fluid, the antibodies are purified using a HiTrap Protein G orHiTrap Protein A column (Pharmacia) as appropriate for theimmunoglobulin subclass and species. Prior to chromotography the ascitesare diluted with an equal volume of 10 mM sodium phosphate, 0.9% NaCl,pH 7.4 (PBS) and clarified by passage through a 0.22 μm filter. Thefiltrate is loaded onto the column in PBS and the column is washed withtwo column volumes of PBS. The antibody is eluted with 100 mMGlycine-HCl, pH 2.7 (for protein G) or 100 mM citric acid, pH 3.0 (forprotein A). The eluate is collected, into {fraction (1/10)} volume 1 MTris-HCl. pH 8.0. The final pH is 7.5. Fractions containing theantibodies are confirmed by SDS-PAGE and then pooled and dialyzedagainst PBS.

[0208] The different samples of purified antibodies are each diluted to100 μg/ml. Each different antibody sample is applied to a separate patchof an array of aminoreactive monolayer patches (see Example 4, above)using a computer-aided, capillary-based microdispensing system. After animmobilization period of 30 minutes at 30° C., the array is rinsedextensively with 10 mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.

[0209] Transformed human cells grown in culture are collected by lowspeed centrifugation, briefly washed with ice-cold PBS, and resuspendedin ice-cold hypotonic buffer containing Dnase/Rnase (10 μg/ml each,final concentration) and a mixture of protease inhibitors. Cells aretransferred to a microcentrifuge tube, allowed to swell for 5 minutes,and lysed by rapid freezing in liquid nitrogen and thawing in ice-coldwater. Cell debris and precipitates are removed by high-speedcentrifugation and the supernatant is cleared by passage through a 0.45μm filter. The cleared lysate is applied to the monoclonal antibodyarray described above and allowed to incubate for 2 hours at 30° C.After binding the array is washed extensively as in Example 9, above.The location and amount of bound proteins are determined by opticaldetection.

[0210] All documents cited in the above specification are hereinincorporated by reference. In addition, the co-pending U.S. patentapplication “Arrays of Protein-Capture Agents and Methods of UseThereof”, filed on Jul. 14, 1999, with the identifier 24406-0006, forthe inventors Peter Wagner, Steffen Nock, Dana Ault-Riche, and ChristianItin, is herein incorporated by reference in its entirety. Variousmodifications and variations of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the art are intended to be within the scope of the following claims.

What is claimed is:
 1. An array of proteins, comprising: (a) asubstrate; (b) at least one organic thinfilm on some or all of thesubstrate surface; and (c) a plurality of patches arranged in discrete,known regions on portions of the substrate surface covered by organicthinfilm, wherein each of said patches comprises a protein immobilizedon the underlying organic thinfilm.
 2. The array of claim 1 whichcomprises at least about 10 of said patches.
 3. The array of claim 2which comprises at least about 100 of said patches.
 4. The array ofclaim 3 which comprises at least about 10³ of said patches.
 5. The arrayof claim 1 which comprises at least about 10 different immobilizedproteins.
 6. The array of claim 5 which comprises at least about 100different immobilized proteins.
 7. The array of claim 6 which comprisesat least about 1000 different immobilized proteins.
 8. The array ofclaim 1, wherein the area of the substrate surface covered by each ofthe patches is no more than about 0.25 mm².
 9. The array of claim 8,wherein the area of the substrate surface covered by each of the patchesis between about 1 μm² and about 10,000 μm².
 10. The array of claim 1,wherein the patches are all contained within an area of about 1 cm² orless on the surface of the substrate.
 11. The array of claim 1, whereinall of the proteins immobilized on the array are functionally related.12. The array of claim 1, wherein all of the proteins immobilized on thearray are structurally related.
 13. The array of claim 1, wherein all ofthe proteins immobilized on the array are members of the same family.14. An array of claim 13, wherein said family is selected from the groupconsisting of growth factor receptors, hormone receptors,neurotransmitter receptors, catecholamine receptors, amino acidderivative receptors, cytokine receptors, extracellular matrixreceptors, antibodies, lectins, cytokines, serpins, proteases, kinases,phosphatases, ras-like GTPases, hydrolases, steroid hormone receptors,transcription factors, heat-shock transcription factors, DNA-bindingproteins, zinc-finger proteins, leucine-zipper proteins, homeodomainproteins, intracellular signal transduction modulators and effectors,apoptosis-related factors, DNA synthesis factors, DNA repair factors,DNA recombination factors, cell-surface antigens, hepatitis C virus(HCV) proteases and HIV proteases.
 15. The array of claim 1, wherein theproteins are antibodies or antibody fragments.
 16. The array of claim 1,wherein the proteins are protein-capture agents.
 17. The array of claim1, wherein the organic thinfilm on the array is less than about 20 nmthick.
 18. The array of claim 1, wherein the organic thinfilm on thearray comprises a monolayer.
 19. The array of claim 18, wherein themonolayer comprises a self-assembled monolayer comprising molecules ofthe formula (X)_(a)R(Y)_(b) wherein R is a spacer, X is a functionalgroup that binds R to the surface, Y is a functional group for bindingthe protein onto the monolayer, and a and b are, independently,integers.
 20. The array of claim 19, wherein both a and b are
 1. 21. Thearray of claim 19, wherein: said substrate is selected from the groupconsisting of silicon, silicon dioxide, indium tin oxide, alumina,glass, and titania; and X, prior to incorporation into said monolayer,is selected from the group consisting of a monohalosilane, dihalosilane,trihalosilane, trichlorosilane, trialkoxysilane, dialkoxysilane,monoalkoxysilane, carboxylic acids, and phosphates.
 22. The array ofclaim 19, wherein the substrate comprises silicon and X is an olefin.23. The array of claim 1, wherein the substrate comprises a polymer. 24.The array of claim 19, further comprising at least one coating betweensaid substrate and said monolayer, wherein said coating is formed on thesubstrate or applied to the substrate.
 25. The array of claim 24,wherein: the coating comprises a noble metal film; and X prior toincorporation into said monolayer, is a functional group selected fromthe group consisting of an asymmetrical or symmetrical disulfide,sulfide, diselenide, selenide, thiol, isonitrile, selenol, trivalentphosphorus compounds, isothiocyanate, isocyanate, xanthanate,thiocarbamate, phosphines, amines, thio acid and dithio acid.
 26. Thearray of claim 24, wherein the coating comprises titania or tantalumoxide and X is a phosphate group.
 27. The array of claim 1, wherein eachprotein is immobilized on the organic thinfilm by an affinity tag.
 28. Abiosensor comprising an array of proteins of claim
 1. 29. Amicromachined device comprising an array of proteins of claim
 1. 30. Adiagnostic device comprising an array of proteins of claim
 1. 31. Amethod for screening a plurality of proteins for their ability tointeract with a component of a sample, comprising: (a) delivering thesample to the array of claim 1 comprising the proteins to be screened;and (b) detecting, either directly or indirectly, for the interaction ofsaid component with the immobilized protein of each patch.
 32. Themethod of claim 31, wherein the component is a protein.
 33. A method forscreening a plurality of proteins for their ability to bind a particularcomponent of a sample, comprising: (a) delivering said sample to thearray of claim 1 comprising the proteins to be screened; and (b)detecting, either directly or indirectly, for the presence or amount ofsaid particular component retained at each patch.
 34. The method ofclaim 33, wherein said particular component is a protein.
 35. The methodof claim 33, further comprising the step: (d) further characterizingsaid particular component retained on at least one patch.
 36. A methodof assaying for protein-protein binding interactions, comprising: (a)delivering a sample comprising at least one protein to be assayed forbinding to the array of claim 1; and (b) detecting, either directly orindirectly, for the presence or amount of the protein from the samplewhich is retained at each patch.
 37. A method of assaying in parallelfor a plurality of analytes in a sample, comprising: (a) delivering thesample to the array of claim 1, wherein at least one of the immobilizedproteins of said array can react with each of said analytes; and (b)detecting for the interaction of the analytes with the immobilizedprotein at each patch.
 38. A method of assaying in parallel for aplurality of analytes in a sample, comprising: (a) delivering the fluidsample to the array of claim 1, wherein at least one of the immobilizedproteins of said array can bind each of said analytes; and (b)detecting, either directly or indirectly, for the presence or amount ofanalyte retained at each patch.
 39. The method of claim 38, furthercomprising the step: (d) further characterizing the analyte retained onat least one patch.